AVOIL Characterisation of the toxicity of aviation turbine ......flights and one for single aisle or...
Transcript of AVOIL Characterisation of the toxicity of aviation turbine ......flights and one for single aisle or...
Final Report EASA_REP_RESEA_2015_2
Research Project:
AVOIL
Characterisation of the toxicity of aviation
turbine engine oils after pyrolysis
EASA.2015.HVP.23: Characterisation of the toxicity of aviation turbine engine oils after pyrolysis (AVOIL)
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Project:
EASA.2015.HVP.23 – “Characterisation of the toxicity of aviation turbine engine oils
after pyrolysis (AVOIL)”
Period:
12.2015 – 02.2017
Date:
Print version February 16th
2017
Authors:
Marc Houtzager B.Sc, (TNO),
John Havermans Ph.D, (TNO),
Daan Noort Ph.D, (TNO),
Marloes Joosen, Ph.D, (TNO),
Jan Bos M.Sc, B.Eng (TNO),
Rob Jongeneel RTE M.Sc, (RIVM),
Petra van Kesteren M.Sc, (RIVM),
Harm Heusinkveld Ph.D, (RIVM),
Irene van Kamp Ph.D, (RIVM),
Sicco Brandsma Ph.D, (IVM),
Remco Westerink Ph.D, (IRAS).
Document Ref:
AVOIL project Nr. 923642 / 060.18709
Version No:
Final print version
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EASA.2015.HVP.23
Characterisation of the toxicity of aviation turbine engine oils after pyrolysis (AVOIL) – Final Report
February 16th 2017
Report Authors:
Marc Houtzager1, John Havermans
1, Daan Noort
1, Marloes Joosen
1, Jan Bos
1, Rob Jongeneel
2, Petra van
Kesteren2, Harm Heusinkveld
2, Irene van Kamp
2, Sicco Brandsma
3, Remco Westerink
4
1. The Netherlands Organization for Applied Scientific Research (TNO).
2. National Institute for Public Health and the Environment (RIVM)
3. Institute for Environmental Studies (IVM)
4. Institute for Risk Assessment Sciences (IRAS)
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Executive Summary
There are concerns among the international governments, pilots, cabin crew and passengers and other
stakeholders of commercial jet aircraft about possible health risks associated with reports of the presence of fumes
in the air supplied to aircraft cabins. One particular area of concern is contamination of the cabin air after pyrolysis
of jet engine fluids, not to forget the additives that are already present in the oil.
The European Aviation Safety Agency (EASA) contracted the consortium of the Netherlands Organisation for
Applied Scientific Research (TNO) and the Dutch National Institute for Public Health and the Environment (RIVM)
in November 2015 to perform a study on the characterization of the toxicity of aviation turbine engine oils after
pyrolysis (AVOIL). This study is registered under the number EASA.2015.C16.
Aim of this work is to characterize the toxic effects of chemical compounds that are released into the cabin or
cockpits of transport aircraft. The characterisation is aimed at the toxic effects of aviation turbine engine oil as a
mixture of compounds, including potential pyrolysis breakdown products. Four tasks have been defined:
Task 1. Performance of scientific literature review and selection of applicable engine/APU oils.
Task 2. Design of a test methodology for the chemical characterization and toxic effects of these oils after
pyrolysis.
Task 3. Performance of the chemical characterization and toxic effects of the oils after pyrolysis.
Task 4. Analysis of the human sensitivity variability factor.
This report describes the performance of the tasks, the obtained results and the drawn conclusions.
The literature descriptive review was focused on the type of effects reported in humans, measurements of oil
components in an aircraft, in vitro and in vivo toxicity tests conducted with engine oil or its fumes, and composition
of aviation engine oil, fumes or pyrolysis products. The findings of this descriptive review were used in the set-up of
the experiments in this study. The literature showed that in time, analytical techniques for dedicated components
that may be present due to the used oil, have been improved significantly. This explains probably the fact, that in
older literature no components as tricresyl phosphate (TCP) were found. Further, it showed that the temperature in
an aircraft engine compartment, where oil vapour and pyrolysis products may be formed, can reach temperatures
above 500°C and therefore more toxic fumes (containing carbon monoxide (CO) and trimethylolpropane phosphate
(TMPP)) may be generated.
Experimental work was performed using two generally used brands of oil: one typically for twin-aisle or long range
flights and one for single aisle or short range flights. The latter one included also operationally used engine oil.
Experimental work consisted of three distinct parts: chemical characterization of oil and oil vapours simulated by
heating oil in combination with purified air, and chemical characterization of oil vapours simulated by heating oil
under pyrolysis conditions. Two flight stages were simulated, i.e. ground level to top of climb and cruise. Finally,
toxic effects were studied using the vapour from pyrolysis of the oil samples.
Chemical characterization of oil and oil vapours by heating oil (under normal air composition)
The experimental set-up of the chemical characterization consisted of heating the oil in different time frames and
adding fresh oil drops at maximum simulation temperature in an emission chamber, simulating the cabin and the
system for sampling.. The experimental set-up shows a good performance for the subsequent experimental work
dedicated to the emission of jet oils at elevated temperatures. The simulation test contained two stages simulating
the start of the engine to top of climb (time frame of the simulation was 30 minutes) and a steady state period for 60
minutes. The simulation tests were kept under controlled and comparable conditions for each oil. At the start of the
simulation test, the temperature of the oils was approx. 21˚C and within 30 minutes the oils reached a temperature
of 350°C. During the next 60 minutes of the simulation test the temperature of the oils reached 375 (± 25)°C. Based
on the chemical characterization of the oils itself and in the emissions of oils performed in the simulation tests for oil
A (new (An) and used (Au)) and oil B (new( Bn), conclusions were established and are presented below.
There was no presence of tri(o,o,o)-cresyl phosphate in the applied oils. It was found that the original oils contained the following isomers: tri(m,m,m,)-, tri(m,m,p)-, tri(m,p,p) and tri(p,p,p)-cresyl phosphate.
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The mass fraction of the TCP isomers found in the oils corresponds with the mass fraction on the fact sheets off
the individual oils.
The simulation test shows that the oils heated at a steady state (375 ± 25°C) emits more TCPs compared to the
simulation whereby the temperature is heated up from 20 to 350°C.
Used oil (Au) showed lower concentrations of TCPs in the simulation test (emission chamber) compared to new oil
(An). Oil An and oil Bn gave comparable results. The simulation test also shows a good correlation between the
composition of TCP isomers found in the original oil compared to the oil vapours at different temperatures.
Comparison of new oil (An) and used oil (Au) shows no significant differences in composition of the four isomers.
It was found that naphthalene was present in the original oil Bn in a concentration of 1.9 mg/kg. The original oil An
did not contain naphthalene as it could not be detected above the detection limit (< 0.58 mg/kg). However, both
anthracene and fluoranthene were found in the applied oils.
By comparison the analytical results of the PAH presence in the vapour and in the original oils, it was found that the
vapour does contain more different types of PAH. Therefore, we suggest the following hypotheses:
- PAHs found in the oil vapour may originate from small concentrations (lower than detection limit) in the original
oil.
- During heating of the oil, a partial oxidation may take place (partial combustion) resulting in the formation of
PAHs due to incomplete combustion.
Based on a selection of the obtained results (aromatics, ketones, and esters) it was found that the emission of both
oils differ at both simulations ‘ground level to top of climb (20 to 350°C) and subsequently cruise speed (375 ±
25°C). For example, the total concentration of aromatic hydrocarbons for oil An and oil Bn at simulation (20 to
350°C) was 36 µg/m3 and 48 µg/m
3 respectively. These concentrations increased drastically (to 1.279 and 3.098
µg/m3 respectively) at cruise simulation (375 ± 25°C). Similar results were observed for other various volatile
compounds.
Relative high concentrations of formaldehyde and acetaldehyde were found in the oil vapours. The highest
concentrations of total aldehydes were found for the new oils in the simulation test (20 to 350°C). However, the
used oil A (Au) showed the highest concentrations at 375± 25°C, indicating that the composition and behaviour of
used oil differs from new oil. It has to be stated, that the sampling of aldehydes was affected by the matrix of oil
vapours, resulting in breakthrough of aldehydes. Based on these observations, it can be concluded that due to
matrix effects, the results of the aldehydes sampled are probably underestimated concentrations, results must be
considered as indicative.
From the start of the simulation test until the oil has reached 180˚C hardly any emission of CO arises. However, it
appears that due to an increase of temperature (180 to 375˚C), CO is formed and emitted due to incomplete
combustion.
It was observed that the mass distribution at room temperature of the original oil An consists of alkane chains
(mineral oil) in the range of C24-C50. After 30 minutes of heating from 20 up to 350°C the composition of mineral oil
in the vapour remains unchanged and is comparable with the mineral oil chains found in the original oil at room
temperature. After heating at 375˚C, small changes in composition of the oil are observed, i.e., an increase of
relative low boiling point components. Additionally, unidentified complex mixtures (UME) are formed beneath the
C24-C50 peaks. The results of the mineral oil analysis for used oil A (Au) gave at room temperature similar results as
were found for new oil A (An). Based on the results, it was found that the mineral oil composition for oil Bn is
comparable with the composition found for oil An. Also for oil Bn the alkane chains are in the range of C24 – C50, with
the exception of one peak found around C21. Additionally the ratio of the alkane chains differs between the two oils.
It is evident that the particle number concentrations (PNCs) are rising as the emissions of oil increases. However,
for all the tests except with the used oil A (Au) a decrease in PNCs was found after reaching 30 - 40 minutes of
heating (350 to 375˚C) despite of adding fresh oil to the reaction chamber.
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Chemical characterization of the oils after pyrolysis
The aim was to identify compounds in three oils under a variety of conditions.
In the basic oil patterns, without heating, a set of TCP isomers and 4-octyl-N-(4-octylphenyl)-benzenamine were
found in all three oils. N-phenyl-1-naphthaleneamine was only found in oil Bn, albeit in low concentrations.
Heating under nitrogen led to an increase in the number of compounds found, and led to the identification of 24
compounds in the vapour, found in all oils. A number of compounds was identified unique for either oil An or oil Bn.
In addition, used oil (Au) appeared to contain newly identified compounds compared to unused oil (An), and a
number of compounds originally present appeared to have disappeared during use in an engine jet. This indicates
that during the lifetime of an oil, substantial changes in composition occur.
As it cannot be excluded that oxygen is present in the jet engine, the effect of oxygen addition during pyrolysis was
investigated. These experiments showed that the presence of oxygen led to combustion of the oils, resulting in a
major increase of the number and amount of compounds.
To permit a safety assessment of compounds originating from jet engine oils, a list of compounds identified under
both nitrogen and oxygen conditions, in all oils and during different flight stages was constructed, resulting in 127
compounds. This list can be used to assess the hazard profile of these compounds using the Classification and
Labelling (C&L) database of the European Chemical Agency (ECHA). As a first step, the harmonised classifications
as well as the main self-classifications by manufacturers are added to the list of chemicals, as presented in
Appendix 6. The classification of a substance is an indication for its toxicity.
The experiments were all performed under atmospheric pressure. In a jet engine the pressures can reach almost
10 bars. This could interfere with the formation and evaporation of organic compounds. On the basis of the physical
appearance of used oil (Au) a combustion of turbine lubrication oil is not likely to occur on a large scale. This would
also result in a thick smoke and a pungent odour in the cabin during flight.
Toxic effects of the oils after pyrolysis
Toxicity of the oil vapours was investigated using an in vitro model of the human lung using an air-liquid-interface
system, combined with an in vitro neuronal network system using primary cortical neurons grown on microelectrode
arrays (MEAs). Concentrations as tested in vitro are within the broad range of concentrations reported in literature
during normal flight conditions based on literature (range from 0.3 ng/m3 to 50 µg/m
3). The results showed that
acute exposure of primary rat cortical cultures to medium containing pyrolysis products derived from engine oil,
following transfer to an air-liquid interface equipped with lung epithelial cells, does not induce significant changes in
neuronal activity. However, a trend towards an increase in neuronal activity was observed at the highest tested
concentration and it cannot be ruled out that higher concentrations may affect neuronal activity. Furthermore,
exposure up to 48h resulted in a decreased neuronal activity and it is therefore possible that effects of pyrolysis
products develop only following more prolonged (i.e. 48h and longer).
Analysis of the human sensitivity variability factor
The possible causes of the large variety in reported health symptoms were elucidated by exploring a) the possible
role of genetic differences in metabolism and detoxification between humans and b) the possible influence of stress
and/or coping strategies that may intensify or trigger health complaints. Differences in sensitivity between humans
for the health effects of certain compounds can be expected for those compounds that rely on cytochrome P450
enzymes for their metabolism. However, the broad range of compounds in the cabin air, in combination with other
stressors, has not been systematically mapped, making it difficult to draw conclusions on the contribution of such
inter-individual genetic differences in metabolism and detoxification on the variety in reported symptoms. In view of
the great variety in symptoms and the lack of specificity, it cannot be ruled out that part of the symptoms cannot be
explained by actual exposure levels. The literature shows that we are dealing with symptoms that are quite
common in the general population and fall within the domain of somatically unexplained physical symptoms.
However, overall statements about a potential higher the prevalence of somatically unexplained physical symptoms
in cabin crew are hard to make because symptoms overlap, estimates are dependent on the definitions used and
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the participants in the study. Differences in coping strategies are well-known factors to enhance stress reactions,
which in their own right can lead to acute health complaints and long-term health effects. Whether or not
occupational conditions are responsible for the reported complaints remains unknown until the complete set of
potential chemical exposures is known, including their exposure levels, resulting internal dose levels, full spectrum
of molecular targets (i.e., all different modes of action) and the related no-effect concentrations.
-o0o-
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Inhoud
1 Introduction .................................................................................................................................................... 18
1.1 Introduction ............................................................................................................................................... 18
1.2 Background of AVOIL ............................................................................................................................... 18
2 Aims and Objectives ..................................................................................................................................... 20
3 Task 1: Descriptive literature review and selection of oils ....................................................................... 22
3.1 Introduction ............................................................................................................................................... 22
3.2 Methodology.............................................................................................................................................. 22
Search profile ...................................................................................................................................... 22 3.2.1
Selection of relevant papers ................................................................................................................ 25 3.2.2
Grey Literature..................................................................................................................................... 25 3.2.3
3.3 Results 26
Reference library ................................................................................................................................. 26 3.3.1
Grey literature ...................................................................................................................................... 27 3.3.2
Google search ..................................................................................................................................... 27 3.3.3
Analysis ............................................................................................................................................... 27 3.3.4
3.4 Main findings and usable lessons learnt ................................................................................................... 28
Analysis subcategory 1: type of effects found in humans. .................................................................. 28 3.4.1
Analysis subcategory 2: measurements of oil compounds in an airplane. ......................................... 35 3.4.2
Analysis subcategory 2: In vitro or in vivo toxicity tests conducted with aviation engine oil or its fumes40 3.4.3
The analysis of subcategory 4: Composition of aviation engine oil, fumes or pyrolysis products. ..... 41 3.4.4
Selection of oils ................................................................................................................................... 49 3.4.5
4 Task 2: Chemical characterisation of jet oil ............................................................................................... 50
4.1 Introduction ............................................................................................................................................... 50
4.2 Design of a test methodology for the chemical characterization of oil vapours ........................................ 50
Design of a simulation test set-up ....................................................................................................... 50 4.2.1
Reaction chamber ............................................................................................................................... 51 4.2.2
Emission chamber ............................................................................................................................... 52 4.2.3
The sampling system .......................................................................................................................... 53 4.2.4
4.3 Performance of the simulation tests ......................................................................................................... 55
Experimental ........................................................................................................................................ 55 4.3.1
Temperature conditions during simulation tests .................................................................................. 56 4.3.2
Experimental settings of the simulation tests ...................................................................................... 57 4.3.3
Selected oils for the simulation tests ................................................................................................... 57 4.3.4
Applied sampling and analytical methods ........................................................................................... 58 4.3.5
4.4 Results and discussion ............................................................................................................................. 59
Temperature and loss of weight during the simulation tests ............................................................... 59 4.4.1
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OPEs in oil and oil vapour ................................................................................................................... 61 4.4.2
PAHs in oil and oil vapour ................................................................................................................... 63 4.4.3
Mineral oil in oil and oil vapour ............................................................................................................ 65 4.4.4
VOCs in oil vapour .............................................................................................................................. 68 4.4.5
Aldehydes in oil vapour ....................................................................................................................... 70 4.4.6
Particle number and carbon monoxide in oil vapours ......................................................................... 71 4.4.7
4.5 Limitations for upscaling of the results ...................................................................................................... 76
4.6 Conclusions............................................................................................................................................... 77
5 Task 3: Chemical characterization of the oils after pyrolysis ................................................................... 80
5.1 Background ............................................................................................................................................... 80
5.2 Methods and materials .............................................................................................................................. 81
Experimental design ............................................................................................................................ 81 5.2.1
Basic profiling of turbine oil with GCxGC-ToF-MS .............................................................................. 81 5.2.2
Simulation of the flight pattern under nitrogen and oxygen conditions ............................................... 81 5.2.3
Analysis of pyrolysis samples with GCxGC-ToF-MS .......................................................................... 82 5.2.4
5.3 Results and discussion ............................................................................................................................. 83
Basic profiling ...................................................................................................................................... 83 5.3.1
Overview of peak analysis and identification following pyrolysis ........................................................ 84 5.3.2
Comparison of oil A and oil B after pyrolysis under nitrogen .............................................................. 85 5.3.3
Comparison of new oil (An) and used oil (Au) after pyrolysis under nitrogen ...................................... 87 5.3.4
FTIR analysis of exhaust air, formed during pyrolysis of oil under nitrogen ....................................... 89 5.3.5
Pyrolysis experiments in the presence of oxygen ............................................................................... 90 5.3.6
FTIR analysis of exhaust air, formed during pyrolysis in the presence of oxygen .............................. 92 5.3.7
5.4 Conclusion ................................................................................................................................................ 93
6 Task 3: Performance of the chemical characterization and toxic effects of the oils after pyrolysis. ... 98
6.1 Introduction ............................................................................................................................................... 98
6.2 In vitro approach – Air Liquid Interface ..................................................................................................... 98
ALI - Introduction ................................................................................................................................. 98 6.2.1
ALI - Methods ...................................................................................................................................... 99 6.2.2
Result ALI .......................................................................................................................................... 101 6.2.3
In vitro approach - microelectrode arrays (MEA) .............................................................................. 103 6.2.4
Introduction ........................................................................................................................................ 103 6.2.5
Methods ............................................................................................................................................. 105 6.2.6
Results ............................................................................................................................................... 106 6.2.7
Conclusions and discussion .............................................................................................................. 108 6.2.8
7 Task 4: Analysis of the human sensitivity variability factor ................................................................... 110
7.1 Introduction ............................................................................................................................................. 110
7.2 Genetic differences in metabolism and detoxification ............................................................................ 110
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7.3 The influence of stress/coping strategies on underlying biological pathways leading to health complaints112
Stress as healthy alarm. .................................................................................................................... 114 7.3.1
Definition of somatically unexplained physical symptoms ................................................................ 114 7.3.2
Prevalence ......................................................................................................................................... 115 7.3.3
7.4 Concluding observations ......................................................................................................................... 117
8 Conclusions ................................................................................................................................................. 118
9 References ................................................................................................................................................... 120
Appendix 1 Selection of additional literature ........................................................................................... 128
Appendix 2 Summarizing the health symptoms reported by cabin crew after exposure to contaminated
bleed air. .................................................................................................................................................. 130
Appendix 3 Order of magnitude of measurements of oil compounds found in a realistic setting in an
aircraft. .................................................................................................................................................. 141
Appendix 4 Identification of various compounds in oil vapours (GC/MS/TD) ...................................... 143
Appendix 5 2D TIC chromatogram of TENAX samples ........................................................................... 146
Appendix 6 Classification of chemicals list ............................................................................................. 148
Appendix 7 Chemical analysis of the vapour generated for the in vitro exposures. ........................... 154
Appendix 8 Abbreviations .......................................................................................................................... 155
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1 Introduction
1.1 Introduction
The European Aviation Safety Agency (EASA) contracted the consortium of the Netherlands Organisation for
Applied Scientific Research (TNO) and the Dutch National Institute for Public Health and the Environment (RIVM)
in November 2015 to perform a study on the characterization of the toxicity of aviation turbine engine oils after
pyrolysis (AVOIL). The AVOIL study is registered under the number EASA.2015.C16.
Sub-contractors of the consortium consists of the lnstitute for Risk Assessment Sciences, an interfaculty research
institute within the medical faculties of Utrecht University and VU Amsterdam lnstitute for Environmental Studies.
For additional advices and the possibility to gain operational knowhow and specific knowledge KLM and ADSE
(Aircraft Development and Systems Engineering) were willing to support the consortium.
1.2 Background of AVOIL
There are concerns among the international governments, pilots, cabin crew and passengers and other
stakeholders of commercial jet aircraft about possible health risks associated with reports of the presence of fumes
in the air supplied to aircraft cabins. One particular area of concern is contamination of the cabin air after pyrolysis
of jet engine fluids, not to forget the additives that are already present in the oil.
Despite the widespread use of jet engines, concerns have been raised over the toxicity of vaporised and pyrolysed
engine fluids and their potential to cause short-and long-term health effects. The need for improved understanding
of this potential source of contaminants as well as the possible influence of other sources of airborne substances
(e.g. chemicals emitted by furnishing materials and ambient (outside) air contaminants) is the prime basis for the
current need for in-depth expertise of the toxicity of these materials during a cabin air contamination (CAC).
The cabin (flight deck and passenger cabin) air supply is controlled by a system called the Environmental Control
System (ECS). The air supply in most commercial jet aircraft is bleed-air from the engines that is drawn from the
compressor stage of the engine into the ECS where the air is conditioned to meet the temperature required for the
cabin/flight deck environment. If seals within the engine are not performing effectively, oil and possibly thermal
degradation products of oil can result in contamination of the bleed air. Besides contaminated bleed air, the ECS
itself and the ducts can also be a secondary source of contaminants.
Several studies have considered air quality in aircraft and have found a variety of differences with those of other
indoor environments, including those of homes and offices. For example, low humidity and reduced pressure can
affect passenger’s well-being and their sensory perception and the high level of occupancy means the passengers
themselves are a notable source of carbon dioxide and a variety of organic chemical compounds. “Fume events”,
while rare, have been reported by passengers and crew and may be associated with elevated exposure to airborne
contaminants, the nature of which will depend upon the source. Laboratory studies have shown that vapours and
aerosols released from heated aircraft oils can contain hazardous substances and therefore may be of concern if
present at sufficient concentration during a ‘fume event’ to cause health effects.
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2 Aims and Objectives
The objective of the study is to characterize the toxic effects of chemical compounds that are released into the
cabin or cockpits of transport aircraft. The characterisation is aimed at the toxic effects of aviation turbine engine oil
as a mixture of compounds, including potential pyrolysis breakdown products. Toxic effects of the mixture will be
characterized at the pulmonary and neuronal level, considering the primary routes of exposures and mode of
toxicity. Additionally, identification of suspected toxic individual compounds is provided to adequately assess inter
individual susceptibility. The overall aim is to integrate these aspects based on already available material and
experimental results, to provide a solid basis for steps towards recommendation of Threshold Limit Values for the
identified chemicals.
The Tender proposal describes the requested tasks in EASA consists of the following tasks:
Task 1. Performance of scientific literature review
Task 2. Selection of applicable engine/APU oils
Task 3. Design of a test methodology for the chemical characterization and toxic effects of these oils after pyrolysis
Task 4. Performance of the above mentioned test
Task 5. Analysis of the human sensitivity variability factor
During the kick-off meeting with EASA on the 10th of December 2015 the approach presented by the consortium
was discussed and no major amendments regarding the methodology were made. The following changes and
comments on the project tasks were suggested by the consortium and agreed: Task 2 - it is suggested to combine
this with Task 1 and Task 3. The oil selection will be included in Task 1, as it is based on available information that
shall be gathered. The conditions of the pyrolysis are defined in Task 3, where the pyrolysis products and the
chemical composition of the oil vapour are analysed.
Due to minor changes mentioned in the above text, the project tasks are as follows:
Task 1. Performance of scientific literature review and selection of applicable engine/APU oils
Task 2. Design of a test methodology for the chemical characterization and toxic effects of these oils after pyrolysis
Task 3. Performance of the chemical characterization and toxic effects of the oils after pyrolysis.
Task 4. Analysis of the human sensitivity variability factor
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3 Task 1: Descriptive literature review and selection of oils
3.1 Introduction
The objective of this task is to conduct an inventory of former studies related to aviation turbine engine oil toxicity.
It was suggested to focus the descriptive literature review on several areas of interest, e.g.:
- Characterization of aviation turbine engine oil, its vapour or pyrolysis products.
- Air cabin measurements during flight operations in airplanes of aviation turbine engine oil vapour or
assumed compounds and pyrolysis products.
- Surveys of health complaints related to potential exposure of individuals to aviation turbine engine oil or its
breakdown products.
- Experimental toxicological studies of aviation turbine engine oil in cell cultures and experimental animals
focussed on neurotoxicity and inhalation toxicity.
- Studies relating to specific toxicity (e.g. neurotoxicity) or a mode of action of aviation turbine engine oil or
its pyrolysis products.
The aim of Task 1 is to perform an analysis of relevant existing studies related to aviation turbine engine oil toxicity
to obtain usable lessons for the other tasks of the overall study. To ensure an optimal inventory of relevant studies,
search profiles need to be as specific as possible. The areas of interest presented above are reformulated into four
subcategories aimed to support the information specialist in determining a search profile. The link of the
subcategory with the other task in this study is described:
1. Type of effects found in humans and possible mechanistic explanations
- This links to Task 4 where an explanatory chapter will be written discussing the various factors that
could explain the variability in health complaints observed
2. Order of magnitude of measurements of oil compounds found in a realistic setting in an airplane
- This links to the outcome of Task 3 where exposures in realistic settings could be compared with
effect levels found in this study
3. In vitro or in vivo toxicity tests conducted with aviation engine oil or its fumes
- This links to Task 3 to identify any usable lessons for this study
4. Composition of aviation engine oil, fumes or pyrolysis products
- This links to Task 2 to characterize the engine oil pyrolysis products
3.2 Methodology
Search profile 3.2.1
An information specialist, located at the RIVM, was asked to compose a search profile for each subcategory based
on the defined subcategories and consultation with a RIVM team member (Table 3.1).
Table 3.1 Search profiles for each category
Category Search profile
1. Type of effects found
in humans and
possible mechanistic
explanations
Medline
Database: MEDLINE 1950 to present, MEDLINE In-Process & Other Non-
Indexed Citations
Search Strategy:
--------------------------------------------------------------------------------
1 (aircrew* or air* crew* or flight crew* or flightcrew* or air* pilot* or cabin
crew* or crewmember* or (air* adj5 passenger*)).tw. (3130)
2 Aircraft/ (7790)
3 Aviation/ (5664)
4 1 or 2 or 3 (14841)
5 inhalation exposure/ or occupational exposure/ (49947)
6 *Environmental exposure/ae (7160)
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7 Air Pollution, Indoor/ae, an [Adverse Effects, Analysis] (7168)
8 *Air Pollution/ae, an [Adverse Effects, Analysis] (6292)
9 (bleed air or cabin air or cockpit air or (contamin* adj air*) or air quality or
exposure or (health adj effect*) or (health adj complaint*) or neurologic* or
neurotox* or (neuro* adj2 tox*)).tw. (888018)
10 Neurotoxicity Syndromes/ or Syndrome/ (111874)
11 5 or 6 or 7 or 8 or 9 or 10 (1020200)
12 cresols/ or tritolyl phosphates/ (4052)
13 (tricresyl phosphate* or tricresylphosphate* or tri-ortho-cresyl phosphate*
or organophosphate*).tw. (8091)
14 (((engine or hydraulic or turbine or lubricat* or jet or pyroly*) adj2 oil) or
(hydraulic adj fluid)).tw. (618)
15 Fuel Oils/ (1261)
16 Oils/ae, po, to [Adverse Effects, Poisoning, Toxicity] (1111)
17 Smoke/ae, to [Adverse Effects, Toxicity] (2666)
18 (fume* or odour* or odor*).tw. (27416)
19 12 or 13 or 14 or 15 or 16 or 17 or 18 (44760)
20 aerotoxic syndrome.tw. (15)
21 4 and 11 and 19 (63)
22 20 or 21 (68)
Scopus
((((TITLE-ABS-KEY((aircrew* OR air*-crew* OR flight-crew* OR flightcrew* OR
air*-pilot* OR cabin-crew* OR crewmember* OR crew-member* OR (air* W/5
passenger*)))) OR (TITLE(aircraft OR aviation))) AND (TITLE-ABS-
KEY((bleed-air OR cabin-air OR cockpit-air OR (contamin* W/1 air*) OR air-
quality OR exposure OR (health W/1 effect*) OR (health W/1 complaint) OR
(adverse-effect*) OR neurologic* OR inhalat* OR (indoor W/1 air) OR
neurotoxic* OR (neuro* W/2 tox*))))) AND ((TITLE-ABS-KEY((tricresyl-
phosphate OR tricresylphosphate OR tri-ortho-cresyl-phosphate OR
organophosphate))) OR (TITLE-ABS-KEY(((engine OR hydraulic OR turbine
OR lubricat* OR jet OR pyroly*) W/2 oil))) OR (TITLE-ABS-KEY((hydraulic W/1
fluid))) OR (TITLE-ABS-KEY((fume OR odour* OR odor* OR neurotox*))))) OR
(TITLE-ABS-KEY(aerotoxic-syndrome))
2. Order of magnitude of measurements of oil compounds found in a realistic setting in an airplane
Database: MEDLINE 1950 to present, MEDLINE In-Process & Other Non-
Indexed Citations
--------------------------------------------------------------------------------
1 (airplane* or aircraft* or aviation or inflight or in-flight or bleed air or cockpit
air).tw. (12587)
2 Aircraft/ (7795)
3 Aviation/ (5665)
4 1 or 2 or 3 (20861)
5 (((engine or hydraulic or turbine or lubricat* or jet or pyroly* or fume*) adj2
oil?) or (hydraulic adj fluid?) or (lubrication adj additive?) or (oil? adj
additive?)).tw. (989)
6 Fuel Oils/ or Oils/ or Lubrication/ (13534)
7 5 or 6 (14286)
8 cresols/ or tritolyl phosphates/ or exp Organophosphates/ (27236)
9 (tricresyl phosphate* or tricresylphosphate* or tri-ortho-cresyl phosphate*
or organophosphate*).tw. (8101)
10 Volatile Organic Compounds/ or Hazardous Substances/ (12057)
11 (volatile organic compound? or semi-volatile organic compound? or
VOC? or SVOC? or hazardous substance? or hazardous compound?).tw.
(9124)
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12 8 or 9 or 10 or 11 (50734)
13 4 and 7 and 12 (36)
14 exp Chemistry Techniques, Analytical/ or Environmental monitoring/
(1920781)
15 (method* or measure* or detect* or spectrometr* or quantif* or
characteri* or investigat*).tw. (9280153)
16 14 or 15 (10097693)
17 13 and 16 (26)
Scopus
TITLE-ABS-KEY(airplane OR aircraft OR aviation OR bleed-air OR cockpit-air)
AND TITLE-ABS-KEY(((engine OR hydraulic OR turbine OR lubricat* OR jet
OR pyroly* or fume*) W/2 oil) OR (hydraulic W/1 fluid) OR (lubrication W/1
additive?) OR (oil W/1 additive?)) AND TITLE-ABS-KEY(tricresyl-phosphate*
OR tricresylphosphate* OR tri-ortho-cresyl-phosphate* OR organophosphate
OR volatile-organic-compound? OR semi-volatile-organic-compound? OR
VOC? OR SVOC?)AND TITLE-ABS-KEY(method* OR measure* OR detect*
OR *spectrometric* OR quantif* OR characteri* OR investigat*)
3. In vitro or in vivo toxicity tests conducted with aviation engine oil or its fumes
Database: MEDLINE 1950 to present, MEDLINE In-Process & Other Non-
Indexed Citations
--------------------------------------------------------------------------------
1 (airplane* or aircraft* or aviation or inflight or in-flight or bleed air or cockpit
air or aerotox*).tw. (12595)
2 Aircraft/ (7795)
3 Aviation/ (5665)
4 1 or 2 or 3 (20865)
5 (((engine or hydraulic or turbine or lubricat* or jet or pyroly* or fume*) adj2
oil?) or (hydraulic adj fluid?) or (lubrication adj additive?) or (oil? adj
additive?)).tw. (989)
6 Fuel Oils/ or Oils/ or Lubrication/ (13534)
7 5 or 6 (14286)
8 (toxicity adj2 test*).tw. (8497)
9 exp Toxicity Tests/ (94283)
10 ((in vitro or in vivo) and (toxic* or neurotoxic* or cytotox* or intoxic* or
test* or study or studies or assay*)).tw. (833926)
11 8 or 9 or 10 (913224)
12 4 and 7 and 11 (13)
Scopus:
TITLE-ABS-KEY(airplane OR aircraft OR aviation OR bleed-air OR cockpit-air
OR aerotox*) AND TITLE-ABS-KEY(((engine OR hydraulic OR turbine OR
lubricat* OR jet OR pyroly* or fume*) W/2 oil) OR (hydraulic W/1 fluid) OR
(lubrica* W/1 additive) OR (oil W/1 additive?)) AND TITLE-ABS-KEY (((in-vitro
OR in-vivo ) AND (*toxic* OR test* OR study OR studies OR assay)) OR
(*toxic* W/2 (test* OR study OR studies OR assay)))
4. Composition of aviation engine oil, fumes or pyrolysis products
Scopus:
(TITLE(aircraft OR aviation OR bleed-air OR cockpit-air OR aeroengine* OR
jet)) AND (TITLE-ABS-KEY(lubricat* OR lubricant* OR hydraulic-fluid* OR
engine-oil* OR jet-oil*)) AND (TITLE(chemical OR composition OR analy* OR
identificat* OR spectromet* OR investigat* OR pyroly* OR characteri* OR
identificat* OR measure* OR monitor* OR assess* OR study OR toxic*))
This search yielded 197, 45, 25 and 167 references respectively per category. A personalized search was
performed by the information specialist in Embase and Toxcenter to obtain additional results not found using the
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abovementioned protocol. This generated 5, 8, 10 and 13 more references respectively. The Endnote files are
provided as separate documents accompanying this report.
Selection of relevant papers 3.2.2
The references found by the information specialist were screened. The first assessment was performed based on
the title. Two scientists, one based at IVM and one based at RIVM, have independently screened all the titles and
decided whether the article is relevant for this literature review or not. Next, the abstract of the selected article was
read and subsequently it was decided whether the article was still relevant. In the end, all articles that were
included by only one researcher were jointly discussed for inclusion or exclusion.
The work presented here is a descriptive literature review describing the key points of each study.
Grey Literature 3.2.3
Besides peer reviewed literature also grey literature reports (non-peer reviewed literature which are not controlled
by commercial publishers) were identified as potential relevant by the consortium (Table 3.2).
Table 3.2. Grey literature identified at the start of the project.
References Title
Crump et al. (2011). Aircraft Cabin Air Sampling Study. Cranfield University, UK, Institute
of Environment and Health. Cranfield Ref No YE29016V
EPAAQ (2011). Contamination of aircraft cabin air by bleed air – a review of the
evidence. Document reviewing evidence up to September 2009.
Adelaide, Australia, Expert Panel on Aircraft Air Quality
Occupational Health Research
Consortium in Aviation
(OHRCA) (2014)
Cabin Air Quality Incidents Project Report
Solbu (2011) Airborne organophosphates in the aviation industry. Sampling
development and occupational exposure measurements.
Dissertation Kasper Flatland Solbu. National Institute of
Occupational Health and University of Oslo, Oslo, Norway
TNO (2013b) Investigation of presence and concentration of tricresyl phosphates
in cockpits of KLM Boeing 737 aircraft during normal operational
conditions. TNO 2013 R11976, 12 December 2013
Committee on toxicity of
chemicals in food consumer
products and the environment
(COT) (2007)
Statement on the review of the cabin air environment, ill-health in
aircraft crews and the possible relationship to smoke/fume events in
aircraft
United Kingdom Parliament
2000
Select Committee on Science and Technology – Fifth Report
United Kingdom Parliament
2007
Select Committee on Science and Technology – First Report
update
Professor Michael Bagshaw.
(2014)
Health Effects of Contaminants in Aircraft Cabin Air. Summary
Report v2.7
German Federal Bureau of
Aircraft Accident Investigation
(2014)
Study of reported occurrences cabin air quality in transport aircraft
IOM (2012) Cabin Air – surface residue study Report
Parliament of the
Commonwealth of Australia
(2000)
Air Safety and Cabin Air Quality in the BAe 146 Aircraft
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During the interim meeting with EASA (23 February 2016) and subsequent correspondence via e-mail additional
grey literature was provided which is summarized in Appendix 1. Some of the reports were already listed in Table
3.2 and therefore excluded from the list. In total 29 documents, links or references were provided and screened for
relevance.
3.3 Results
Reference library 3.3.1
The 260 references retrieved by the information specialist for category 1 and 2 were screened on title and abstract.
In total 28 references were directly included and three references were jointly discussed (see Table 3.3). This
resulted in a total of 28 papers; full text articles were obtained from 26 articles. For two articles no full text could be
retrieved by the consortium without substantial costs. The matching endnote file is provided as a separate file
accompanying this report.
Table 3.3. Selection process of the retrieved references
Category-1
Category-1
extraa Category-2
Category-2
extraa
Total references 202 5 45 8
Reviewer I II I II I II I II
Not selected 133 118 5 5 29 27 4 4
Selected by title 69 84 2 2 16 18 4 4
Selected after summary 20 21 0 0 7 7 1 1 a A personalized search was performed by the information specialist in Embase and Toxcenter to obtain additional
results not found using the abovementioned protocol.
The 215 references retrieved by the information specialist for category 3 and 4 were also screened on title and
abstract. In total 28 references were directly included and 17 references were jointly discussed (see Table 3.4).
This resulted in a total of 38 papers; full text articles were obtained from 20 articles. For 18 articles, mostly
conference papers, no full text could be retrieved by the consortium without substantial costs. The matching
endnote file is provided as a separate file accompanying this report.
Table 3.4. Selection process of the retrieved references
Category-3
Category-3
extraa Category-4
Category-4
extraa
Total references 25 10 167 13
Reviewer I II I II I II I II
Not selected 21 14 2 1 130 123 9 7
Selected by title 4 11 8 9 37 44 4 6
Selected after summary 3 10 8 9 14 23 3 5 a A personalized search was performed by the information specialist in Embase and Toxcenter to obtain additional
results not found using the abovementioned protocol.
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Grey literature 3.3.2
Beside the scientific papers, several reports provided by the EASA and consortium members listed in Table 3.2
and Appendix 1 were considered relevant for the literature review of category 1 and 2. The justification for the
selection is given in Table 3.2 and Appendix 1. The report from EPAAQ (2011): Contamination of aircraft cabin air
by bleed air – a review of the evidence. Document evidence up to September 2009 is regarded most relevant
containing useful information for subcategory 3 and 4 and was included in the analysis.
Literature provided by EASA and consortium members: in total four reports are considered relevant for subcategory
3 and 4 and included in the analysis. The justification for the selection is provided in Appendix 1.
Google search 3.3.3
To screen for any missing relevant document related to subcategory 4, a Google search was performed with the
following keyword:
Search 1: Pyrolysis, jet engine oil, air quality Search 2: Pyrolysis, turbine oil, cabin air Search 3: Fume event, engine oil, contaminated air The first 30 hits of each of the three google searches were screened for relevant information. The scientific papers
and reports that were included so far were selected by title and abstract. The following 3 scientific paper/reports
were selected and discussed between the reviewers:
1. Michaelis, S.. Contaminated aircraft cabin air. Journal of biological Physics and Chemistry, vol 11,
2011, p 132-145 (Michaelis, 2011)
2. Cabin air quality Civil Aviation Authority (CAA) ISBN 0 86039 961 3. Published February 2004
(https://publicapps.caa.co.uk/docs/33/CAPAP2004_04.PDF)(CAA, 2004)
3. Hildre, T. T., Jensen, J. K., Fume Events in Aircraft Cabins. Master thesis, NTNU, Trondheim, June
2015.
(http://brage.bibsys.no/xmlui/bitstream/handle/11250/2353003/12960_FULLTEXT.pdf?sequence=1&is
Allowed=y) (Hildre and Jensen, 2015)
After discussing these results, it was decided to include these three paper/reports in the analysis for subcategory 4.
Analysis 3.3.4
For category 1 and 2 an extensive meta-analysis of the reported symptoms or substance concentrations is not
foreseen, instead a table is generated with descriptive elements (type of study, number of participants or
measurement, symptoms or type of substance and study conclusions) of the selected studies.
For category 3 and 4, the descriptive review was focussed on information considered useful for the experimental
set-up in the other tasks of this project. These include the generation of vapours and pyrolysis products at relevant
conditions. In order to extract useful lessons for these experiments, the analysis of the selected articles was
focussed on those topics deemed useful for next steps in the study.
The coordinators of these experiments were requested to provide specific subjects that shall be taken into account
in the literature review and that may contribute to their experimental set-up.
Summarized, information was requested on: • The temperature in an aircraft engine compartment where oil vapours and pyrolysis products may be
formed.
• Any specifics on the conditions during a fume event, e.g. temperature, pressure, amount of oxygen. This can refer to the conditions in the engine compartment where vapours and pyrolysis products may be formed, but also to conditions in the cabin where people may be exposed.
• The type of products that may be formed during the heating and/or pyrolysis process, e.g. vapours, aerosols.
• Any indications on the time period of the pyrolysis. • In vitro experiments performed with vapours or pyrolysis products of aviation engine oils.
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It is noted that information on these subjects by the experiments coordinators was also obtained via other
information routes, including own literature databases and information obtained from ADSE.
3.4 Main findings and usable lessons learnt
Analysis subcategory 1: type of effects found in humans. 3.4.1
The 19 selected papers were screened for effects found in humans exposed to “contaminated” aircraft cabin air.
The symptoms found were summarized in the Table 3.5 as well as study design, data set and conclusion of the
study.
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Table 3.5. Study design, information regarding the data set studies, and general conclusions according to the
authors were summarized for each study.
Study design Data set Symptoms Conclusiona Reference
This study presents
the exposure of
Westland Sea King
helicopter crew to
engine exhaust
fumes, clinical signs
of CO intoxication,
and levels of
carboxyhemoglobin
saturation (SpCO)
after standard
operation training
flights.
69 completed surveys
of crew specialists
pilots (n=18), system
operators (n=14),
engineers (n=11),
rescuers (n=11), flight
physicians (n=15)
were collected over a
2 week period.
The median duration of
the training session was
80 minutes. 64% reported
subjective exposure to
engine exhaust during the
training sessions. 8.6%
reported clinical symptoms
such as; exhaustion (n=4),
headache (n=1) and
nausea (n=1).
Exposure to engine exhaust
fumes in common, whereby
8.6% reported clinical symptoms
mainly during open cargo door
operations. Toxic SpCO levels
were not reached however, 29%
showed SpCO levels outside the
normal range (>4%).
Busch (2015)
General practitioner
(GP) M. Somers has
seen 39 flight crew
members (7 pilots
and 32 flight
attendants) and one
passenger who
reported symptoms
in relation to
exposure to fumes
in the cabin of
mainly the BAe 146.
In this study, data files
were collected over a
period of 6 years from
36 flight crew
members who came to
see GP M. Somers
because of their health
concerns regarding to
the exposure to
contaminated cabin
air.
The most common
symptoms observed after
long and short-term
exposure where: nausea,
headache, mucous
membrane irritation,
lethargy and cognitive
dysfunction. Cognitive
impairment were also
reported such as unable to
speak, poor memory, felt
drugged, unable to think
clearly, disorientation, trips
over words, couldn’t put
dates in order etc. All
symptoms reported are
listed in Table A.1 in
Appendix 2.
Symptoms were reported during
taxiing, take-off, climbing, top of
descent, descending and
landing. Correlation between
fume events and technical faults
were observed. Overall there is
a need for a reporting system
that is objective and
independent of the operator.
Exposure to contaminated cabin
air has influences on the
worker’s health, finances, future
work capacity and may also
affect the safety of the aircrew
and passengers.
Somers (2005)
In this
epidemiological
study, the health
effects of aircrew
members were
investigated through
a questionnaire
survey. The aircrew
monitored were
flying with the BAe
146 and/or A320
aircraft.
The collected data set
consisted of 50
Australian aircrew
members (72%
female) in the age
range of 26 to 59
years. 70% of the
respondents were
cabin crew and 30%
flight crew.
Adverse health symptoms
related to exposure to oil
fumes or odours were
reported by 94% of the
respondents. 96%
reported adverse
symptoms immediately
after flying whereby 74%
also experienced
symptoms for at least 6
months after exposure.
Symptoms reported by the
50 corresponding aircrew
were related to eyes
(76%) and skin irritation
(58%), nausea (58%),
neuropsychological
symptoms including
intense headache (86%),
dizziness and
disorientation (72%), and
exhaustion(78%) etc. All
symptoms reported are
listed in Figures A.1-A.7 of
Appendix 2.
The symptoms reported by the
50 Australian aircrew were
compared to 18 US aircrew and
showed significant similarity in
symptoms.
This study showed that
contaminated cabin air can
result in adverse health effects.
Further investigation regarding
the finding of neurological
impairment, respiratory system
effects, reproductive dysfunction
and long-term effect is needed
according to Winder et al..
According to the authors of the
study, there is still an issue on
reporting health symptoms by
aircrew; they are worried about
job security after filling a
complaint.
Winder et al. (2002b)
This study
investigated a self-
selected group of
affected commercial
aircrew and
document their
symptoms and
treatment in relation
The aircrew members
studied consisted of
39 pilots and 19 flight
attendants. 51% of the
respondents works in
the UK, 37% in
Australia, 10% in the
US and 2% in Egypt.
The most common
symptoms reported by the
aircrew were: neurological
symptoms (impaired
concentration, dizziness,
difficulty thinking, altered
depth perception) followed
by headache, fatigue and
Exposure events were most
frequently detected in the BAe
146 aircraft and during take-off
and ascent. Aircrew reported
that health problems were
underestimated and
undertreated. According to the
author of the study, there is still
Harper (2005)
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Study design Data set Symptoms Conclusiona Reference
to contaminated
cabin air.
mucous membrane
irritation. For more than
50% of the respondents
the symptoms became
chronic and persistent for
months or years. All
symptoms reported are
listed in Figure A.8 of
Appendix 2.
an issue on reporting health
symptoms by aircrew they are
worried about job security after
filling a complaint. In general,
the symptoms occur with odour
or fume event.
In this study the
health symptoms in
aircrew were
investigated while
flying on the BAe
146 aircraft.
21 flight crew
members were
included in this survey
consisting of 19 pilots
and 2 flight attendants.
The responded were
in the age range of 30-
50 years. 20% of the
responded crew were
female. All pilots had
more than 500 flight
hours per year.
A wide range of symptoms
were reported such as
headaches, irritation and
respiratory problems and
disorientation. However
also coordination or
memory effects were
reported which could
influence the flying safety.
Most respondents
considered that the
symptoms were related to
flying with the BAe 146
aircraft. All symptoms
reported are listed in Table
A.2 of Appendix 2.
Due to small sample size and
removal of questionnaires by
one of the airlines, this survey
cannot be considered
representative. However the
small data set does report health
problems by aircrew members
which may be related to the
contaminated air in the BAe 146
aircraft.
Cox and Michaelis (2002)
A survey of health
symptoms in pilots
from the British
Airline Pilots
Association
(BALPA) flying with
the Boeing 737, 757
and the airbus A320.
From the 600
questionnaires that
were sent out, 106
pilots responded. 104
of the 106
respondents were
male.
96 respondents reported
that they experienced
smoke or fume smell
during flights. 93 of the
B757 pilots believed that
the fumes were caused by
oil contamination of the air
supply. The most common
symptoms reported where
irritation, headaches and
fatigue, followed by
confusion, memory
impairment, diarrhoea and
nausea. Improved health
was observed after duty or
on days off. All symptoms
reported are listed in
Figure A.11,Appendix 2.
According to the author of the
study, leak events occur and are
underreported by pilots.
According to the author, this
study shows that contaminated
cabin air causes toxic exposure
and adverse health effect in
aircrew.
Michaelis (2003)
In this study a
neuropsychological
assessment was
performed using a
battery of tests on
aircrew members
that were exposed
to jet oil emission
while flying on the
BAe 146 aircraft.
Neuropsychological
assessment was
carried out on 8 BAe
146 aircraft crew
members who had
been exposed to
engine oil emission.
All aircrew members
were female in the age
range of 24-56 years
who flew on the BAe
146 for 2 to 12 years.
Significant impairment was
observed on test of
reaction time, information
processing speed and fine
motor skills. These
findings could indicate a
serious aviation safety
problem.
Neuropsychological impairment
has been observed on a number
of neuropsychological measures
by the BAe 146 aircrew who
have been exposed to engine
fumes. However, due the small
sample size and no control
group of individuals workers in
the same field not exposed to
BAe 146 jet engine oil emission,
no strong conclusions could be
drawn. Based on the findings the
author would recommend to
conduct a wider-scale study of
BAe 146 aircrew.
Coxon (2002)
This study
investigated the
incapacitation of an
aircraft pilot on a
military C-130A
aircraft after
exposure to
aerosolized or
vaporized engine oil.
A 34 years male pilot
in good health,
observed adverse
health effects while
flying a military C-
130A aircraft.
The following symptoms
were observed by the
pilot: headache, slight
dizziness, nausea,
vomiting, incoordination,
and diaphoresis. 80 min
after the first symptoms he
was examined in the
hospital. He was observed
After exposure to vaporized
aerosolized or vaporized engine
oil a 34 years pilot on a military
C-130A aircraft observed
neurological impairment and
gastrointestinal distress. His
clinical status returned to normal
within 24 hr. Further
investigation into the potential
Montgomery et al. (1977)
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Study design Data set Symptoms Conclusiona Reference
to be lethargic, mild
dysarthria and had
depressed deep tendons
reflexes and anisocoria
(unequal size of the eyes'
pupils).
hazards from exposure to jet
engine oils is highly
recommended.
The goal of this
study was to raise
awareness among
physicians of short
and long term health
effects that may
appear after
exposure to
pyrolyzed engine oil.
Question: are the
symptoms observed
a psychosomatic
disorder or
neurological injury?
A single case study
was used, a middle
aged pilot who had
been flying for 11
years on a particular
type of airplane, to
illustrate some of the
issues regarding
causation and
diagnosis.
The symptoms observed
by the pilot were cognitive
impairment for nine-month
and a 10 years history of
skin ulcerations and
gastrointestinal problems.
During his flights he often
smelt oily fumes which he
supposed to find normal
for this type of airplane.
General practitioner, a
dermatologist and a surgeon
were not able to diagnose the
cause of pilot symptoms. The
pilot license was taken by the
aviation authority on psychiatric
grounds, however the pilot was
not suffering from mood disorder
and has never been examined
by a psychiatrist. After
neurological examination, it was
most likely that the symptoms of
the pilot were related to
exposure to engine oil fumes.
According to the authors, medial
protocols should be created to
prevent misdiagnosis of aircrew.
Mackenzie Ross et al.
(2006)
The study includes a
physical
examination,
neuropsychological
examination and a
PET-scan of the
brain of flight
attendants.
26 North American
airline attendants who
were exposed to toxic
fumes, emanated from
the Auxiliary Power
Unit (APU) to the
aircraft cabin air.
Neurological abnormalities
were detected in 58% of
the flight attendants.
Cognitive impairment such
as impaired balance and
coordination and
movement disorder was
observed. 12 of the 26
attendants were subjected
to PET functional brain
scan and abnormalities
were observed such as
occurrence of
hypofrontality (decrease
frontal and increase
posterior brain function),
and increased function in
some limbic areas,
especially the extended
amygdala region. All
symptoms reported are
listed in Table A.4 in
Appendix 2.
According to the author, the
neurological abnormalities
observed in the studies groups
were most certainly caused by
the exposure to chemical fumes.
The symptoms observed by the
attendants were often
misdiagnosis by physicians.
Therefore, medial protocols
should be created to prevent
misdiagnosis.
Heuser et al. (2005)
This study
investigated the
health complaints by
flight crew as well as
the finding regarding
air quality
measurements
taken during test
flight conditions on
two BAe 146 aircraft
that experienced oil
seal failures.
The findings were
compared with two
BAe 146 aircraft and
a Dash 8 aircraft
that never had been
associated with any
complaints.
Health symptoms
were reported over a
period of 4 months
involving 5 airplanes,
35 flight, 112
individuals of a total of
200 individuals
reported symptoms.
Symptoms reported by the
112 flight crew were
mainly burning trout
(n=48), headache (n=29),
burning eyes (n=27),
disorientation (n=16). The
crew also complained
about sharp odour in
cabin, assault by toxic
fumes, heavy exhaust
smell, de-icing smell,
soapy smell, detergent
smell, dirty sock smell
during take-off, oven
cleaner smell, peroxide
smell, acrid noxious fumes
filling cabin on descent,
aircraft filled with heavy
blue haze, and strong
smoke odor.
All symptoms reported are
Most of the symptoms resolve
within 24 hours. The oil smell in
the BAe 146 aircraft was caused
by oil leaking by one of the
seals. The reported symptoms
headache, nausea,
disorientation could be caused
by low levels of CO. Other
symptoms like, burning eyes and
throat, watery eyes and sinus
congestion, could be caused by
smoke or VOCs. Whereas
neurological symptoms could be
caused by hexane, octane or
neurotoxic additives present in
the engine oil like TCP isomers.
However, no TCP isomers were
detected in the air above the
LOD of 80 μg/m3.
Van Netten (1998)
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Study design Data set Symptoms Conclusiona Reference
listed in Table A.5 in
Appendix 2.
Evaluation of an
odour incident in the
cockpit of an Airbus
A319 passengers
aircraft in relation to
‘aerotoxic
syndrome’.
The evaluation was
based on two pilots
who reported an odour
incident in the cockpit
and were flying further
using oxygen masks.
Both pilots reported
nausea and progressive
cognitive deficits. The pilot
and co-pilot were sent to
the hospital and after 2
hours they were sent
home again. After the
incident the pilot did start
working again after 5
days. However the co-pilot
did not work for 7 months
and was diagnosed with a
post-traumatic stress
disorder.
The odour incident was
not reported as a fume
event.
The German “Bundesstelle fur
The German “Bundesstelle fur
“Fluguntersuchungen” was not
able to provide evidence of
toxins in the cabin air based on
toxicological studies of the
incident. According to the
authors, systematic investigation
on health effect in relation the
contaminated cabin air is
lacking. Such investigation is
needed to find scientifically
evidence that there is a relation
between contaminated cabin air
and adverse health effect.
Schwarzer et al. (2014)
Case study, analysis
of reported
contaminated air
events at one major
US airline over a
period of two years
involving 8 types of
airplanes (A319,
A320, A321, B737,
B757, B767 and
E190.
The focus of this study
was on oil fume
events that were
reported by the
aircrew over a period
of 2 years.
Only some of the
reported events
involved also hydraulic
fluid fume events.
Exposure related to
de-icing fluid, exhaust
fumes and
malfunctioning galley
equipment were
excluded from this
study.
87 fume events were
reported on 47 aircraft
fleet wide. In all 8 types of
aircraft fume events were
reported. However, the
A319, B767 and E190
appeared to be
overrepresented. The
most common inflight
symptoms were
headache, nausea,
coughing and
disorientation. The most
post-flight symptoms were
difficulties to concentrate
and problems with word
recall, fatigue, headache
and memory deficits.
Some of the pilots
developed chronic
neurological symptoms
post-flight and lost their
license. In 83 of the
reported events unusual
odour was reported
described as “dirty socks”.
On 41 of the 87 events
mechanical records confirmed
that oil contaminated the air
supply. In 68 of the 87 reported
events flight attendants reported
symptoms and in almost half of
those events the symptoms
were so serious that emergency
medical care was needed. Flight
attendants were less protected
to fume events than pilots: they
are trained to use oxygen masks
if events occur, flight attendants
are not trained for that.
According to the author, air crew
need to be better trained to
recognize and respond to events
and maintenance workers need
to be better trained to identify
and solve the problem. Filters
and/or monitoring equipment are
needed to detect events and
prevent them.
Murawski et al. (2011)
In this study
symptoms from
seven case studies,
from aircrew in four
airlines operating in
four countries and in
three aircraft models
(B747, Fokker 100,
BAe 146) were
investigated.
Seven case studies
consisting of aircrew
from four airlines
operating in four
countries and in three
aircraft models.
A broad range of
symptoms have been
reported by the aircrew
after exposure to
contaminated cabin air.
Symptoms from single or
short term exposure: 1)
neurotoxic symptoms such
as tunnel vision,
disorientation, shaking,
loss of balance. 2)
neuropsychological
symptoms, memory
impartment, headache,
confusion and feeling
intoxicated. 3)
gastrointestinal symptoms.
4) cardiovascular
symptoms. (5) irritation of
eyes, nose and upper
airways.
Symptoms from long term
According to the authors,
exposure to fume event is
known to be toxic and if
happened to pilots it could
influence the flight safety.
According to the authors factual
evidence is available that
aircrew and passengers can be
directly exposed to airborne
chemicals on aircraft in sufficient
concentration to cause acute,
immediate to long-term
symptoms.
Winder and Balouet
(2001)
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Study design Data set Symptoms Conclusiona Reference
low level exposure: 1)
neurotoxic symptoms,
paresthesia, numbness
fingers, lips and limps. 2)
neuropsychological
symptoms, memory
impairment, lack of
coordination. 3)
gastrointestinal symptoms,
4) respiratory symptoms,
breathing difficulties. 5)
cardiovascular symptoms.
6) skin symptoms. 7)
irritation of eyes, nose and
upper airways. 8)
sensitivity,
immunosuppression,
multiple chemical
sensitivity. 9) general,
weakness and fatigue. All
symptoms reported are
listed in Table A.6 in
Appendix 2.
Summary of aircraft
air quality incident,
symptoms,
exposures and
possible solutions.
Symptoms were
reported by aircrew
members flying for an
unreported airline
company on a MD-80
aircraft and by aircrew
from another
unreported airline
company flying on a
mixed fleet of aircraft.
Exposure to contaminated
bleed air varied within an
aircraft due the differences
of air supply in the cockpit
(100% fresh bleed air)
compared to the flight
deck (60/40
recirculated/fresh bleed
air).
Central nervous system
symptoms were
predominantly followed by
gastrointestinal and
respiratory system
problems.
According to the author, most of
the aircraft air quality incidents
can be traced to contamination
of the bleed air with jet engine oil
and/or hydraulic fluid. The
symptoms reported by the
aircrew may be related to CO
and TCP. Long term chronical
effect are difficult to trace back
to exposure events. Because the
symptoms are a results of
exposure to low levels of
contaminated bleed air for a long
period of time.
Van Netten (2005)
This study
investigated if there
is a link between
exposure to
contaminated bleed
air and
neuropsychological
impairment.
Comparing an
“exposed” group
flying on the BAe
146 and B757 to an
“unexposed” group
flying on the B737.
Questionnaires were
sent to 1500 pilots;
only 70 questionnaires
were returned.
Randomly 29 pilots
were asked to
undergo
neuropsychological
assessment. 15 pilots
were in the exposed
group flying the BAe
146 and B757 and 14
pilots were in the
unexposed group
flying the B737.
Physical and neurological
symptoms were identified
among airline pilots.
Despite the weaknesses
(exposed versus
unexposed by air type
flown and relative small
sample size) the findings
warrant further
investigation.
No significant differences were
identified between the two
groups on the
neuropsychological tests or
mood state measures. However,
it was observed that the air type
flown is not a reliable indicator of
exposure history because in
both groups multiple fume
events have been reported.
The profile of the cognitive
performance was different than
that of an normal population and
comparable to that of framers
exposed to organophosphates.
Cognitive performances are
showing dips in test on attention,
psychomotor speed and visual
sequencing.
Mackenzie Ross et al.
(2006)
This study
investigated
cognitive impairment
in aircrew by an
extensive
neuropsychological
test battery, and if a
neurobiological
substrate could be
found for their
12 aircrew members
(10 male, average age
of 44, 8130 flight
hours) compared to
matching control
group consisting of 11
race car drivers (10
male, average age of
43 years, 233 flight
hours).
The aircrew groups
reported complaints
regarding cognitive
impairment.
Aircrew reported significant
more self-reported cognitive
complaints and depressive
symptoms compared to the
control group. However, the
differences did not reach
statistical significance. Small
brain regions in which brain
white matter microstructures
were affected and higher
Reneman et al. (2015)
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Study design Data set Symptoms Conclusiona Reference
complaints by using
MRI techniques.
cerebral perfusion values in the
left occipital cortex were
observed. The extent of
cognitive impairment was
strongly associated with white
matter integrity, but extent of
estimated number of flight hours
was not associated with
cognitive impairment nor with
reductions in white matter
microstructure.
5 ‘aerotoxic
syndrome’ cases
documented by the
Federal Institute for
Risk Assessment
(BfR) were
investigated
regarding possible
risks for fume events
associated with
TCP-contamination
cabin air.
5 ‘aerotoxic syndrome’
cases in Germany.
Health impairment
however no specific
symptoms are reported.
In none of the 5 ‘aerotoxic
syndrome’ cases studied,
documented by the Federal
Institute for Risk Assessment
(BfR), causality between
possible inhalation exposure and
the health impairment that
occurred was found. According
to the authors, TCP poisoning is
unlikely.
Hahn et al. (2013)
This study
investigated the
association between
neurological deficits
and elevated levels
of autoantibodies in
flight crew.
12 healthy controls
were compared with
34 flight crew (pilot
and attendants) who
experienced adverse
health effects after
exposure to bleed air
and had medical
attention.
Also 1 case study of a
single pilot was
included who was
followed for 21 months
and suffered health
problems after
exposure to
contaminated cabin
air.
The most common
symptoms reported by the
34 aircrew members were
memory deficits (78%),
headaches (62%), fatigue
(53%), muscles weakness
(42%) and imbalance
(35%).
All symptoms reported are
listed in Figure A.9 in
Appendix 2.
The limitation of this study was
the small sample size and the
lack of identification and
quantification of the chemicals to
which the flight crew were
exposed to. However, according
to the authors this study
supports an association between
self-reported neurologic deficits
and levels of autoantibodies
against neuro- and glia-specific
proteins in sera form 34 flight
crew member compared to a
control group.
Development of biomarkers as
reported in this study may help
to detect chemical-induced
central nervous system injury.
Abou-Donia et al. (2013)
A health survey on
4011 flight
attendants from two
airline companies
was compared to
data from a health
survey of a general
population (data
used from the
National Health and
Nutrition
Examination
Surveys (NHANES)
Addresses were
randomly chosen from
union provided lists of
flight attendants.
The mean age of the
flight attendants were
47 years. 20% was
male. 41% had more
than 20 years of
working experiences.
9% were current
smokers and 22-30%
were former smokers.
The largest number of
work related injuries were
musculoskeletal (33%),
respiratory (23%),
neurological problem
(17%), psychological
problems (14%).
Significantly elevated
standardized prevalence
ratios (SPR) were
observed for chronic
bronchitis, cardiac
disease, diagnosed sleep
disorders, fatigue and
depression for the flight
attendants compared to
the general population.
Health symptoms reported
by the flight attendants are
listed in Table A.7 in
Appendix 2.
Almost 50% of the flight
attendants reported one or more
work related injuries. This
compared to 4.2% for all
industries and 10.2% for the
transportation (BLS statistics).
OHRCA (2014)
This study measures
residues on the
internal surfaces of
aircraft and control
environment to
86 wipe samples were
collected using
ethanol-moistened
glass fibre filters. Wipe
samples were
The mean levels observed
in the aircraft ranged up to
3 x 104 ng/m
2 for TCP, to
106 for butyl diphenyl
phosphate (BDPP), to 7 x
Estimated maximum airborne
concentrations were calculated
for tri (o,o,o)-cresyl phosphate
(0.001-0.0006 μg/m3) and TnBP
(10-40 μg/m3). The levels are in
IOM report (2012)
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further investigate
fume events.
collected from 6 sites,
consisting of 4 airports
and 2 control sites;
divided over 5 types of
airplanes, 2 types of
vehicles and 2 offices.
105 for DBPP and to 9 x
104 for TnBP.
Tri-n-butyl phosphate
(TnBP), BDPP and DBPPb
were in general higher in
the wipes collected in the
cockpit compared to the
passengers area. The
levels of TnBP, BDP and
DBPP were also higher in
wipes from aircraft and
airport based vehicles
than in offices. The TCP
levels were higher in
planes than in other
places, with may indicate
that TCP originate from
aircraft sources.
line with those reported in
literature.
This study is based
on investigation and
prevention of
accidents and
incidents, which
have been reported
by the German
Federal Bureau of
Aircraft Accidents
Investigation (BFU)
between 2006-2013.
The goal of this
study was to prevent
future accidents and
incidents.
663 fume events are
investigated in this
report, in 460 cases
smell and in 188
cases smoke
developed. In 180
reports health
impairment was
reported. However, in
only 15 cases health
impairments may
possibly have a
conjunction with cabin
air quality.
Health impairment
reported by 66 pilots were
light-headedness (n=1),
tremor of hands (n=1),
nausea (n=3), eye irritation
(n=6), headache (n=7),
dizziness (n=8), other
(n=8) and multiple (n=32).
Health impairment
reported by 105 cabin
crew were: light-
headedness (n=1), eye
irritation (n=4), nausea
(n=6), dizziness (n=6),
others (n=7) headache
(n=17) and multiple
(n=64).
Heath impairment reported
by 21 passengers were:
dizziness (n=1), headache
(n=1), nausea (n=5), other
(n=6) and multiple (n=8).
Fume events occurred and
resulted in contaminated cabin
air.
According to the authors,
contaminated cabin air has led
to health impairments in
occupants and cabin crew.
Identification of toxic compounds
(such as TCP) in the cabin air
was not performed in the fume
events the BFU investigated.
German Federal Bureau of
Aircraft Accident
Investigation (2014)
a NB: The conclusions were reported by the authors from each study; these conclusions were not reviewed or
evaluated by the authors of this report. b TBP: tri-n-butyl phosphate, BDPP: butyl diphenylphosphate, DBPP: dibutyl phenyl phosphate. Analysis of
subcategory 2: measurements of oil compounds in an airplane.
Analysis subcategory 2: measurements of oil compounds in an airplane. 3.4.2
The 9 selected papers were intensively screened for compounds found in realistic settings in an airplane and
summarized in the Table 3.6.
Table 3.6. Study design, information regarding the data set studies, and general conclusions by the authors of the
studies were summarized for each paper.
Study design Data set Concentrations Conclusion Reference
In this study
methods were
developed to
measure the
exposure to
organophosphates
4 airline companies in
Norway were included
in this study. In total
40 aircraft were
sampled consisting of
jet engine airplanes,
95 within-day OP samples
were collected from cabin
air in 47 flights (jet
airplanes, propeller
airplanes and helicopters).
TCP1 (sum of tri(m,m,m,)-
TCP levels were an order of
magnitude higher in the air
samples collected from the cabin
during a ground experiment on
an airplane that experienced
turbine oil leakage compared to
Solbu et al. (2011)
1 TCP comprises 10 isomers, based the arrangement of the three cresyl groups (ortho, meta or para arrangement).
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Study design Data set Concentrations Conclusion Reference
(OPs) from jet
engine oils and
hydraulic fluids
among aircrew
members.
The sampling
methods included
within-day air
sampling for OPs
using Chromosorb
106, and VOCs
using Tenax-A 60/80
mesh as well as
passive long term
methods by
deposition of OPs
using wipe surface
area and activated
charcoal cloths
(ACC). All samples
were collected under
normal flight
conditions.
propeller airplanes
and helicopters. The
samples were
collected in the cockpit
and the passengers
cabin.
cresyl phosphate,
tri(m,m,p)-cresyl
phosphate, tri(m,m,p)-
cresyl phosphate and
tri(p,p,p)-cresyl phosphate
was only detected in 4% of
all within-day samples,
only in propeller airplanes,
with levels range from <75
ng/m3 to 290 ng/m
3. No
ortho-isomers of TCP
were detected. Triphenyl
phosphate was only
detected in one propeller
airplane with a
concentration of 110
ng/m3. TnBP was detected
in all jet engine and
propeller airplanes and in
58% of the helicopters
sampled with levels range
from 24 to 4100 ng/m3.
BDPP was only detected
in the jet engine and
propeller airplanes with
levels range from <75 to
310 ng/m3.
Passive long term
sampling was only
performed in jet engine
and propeller airplanes
using wipe (n=56) and
ACC (n=56). The OP
levels detected were
summarized in Table A.8
of Appendix 3. Overall, in
the wipes the TCP levels
range from <0.05 to 8.3
ng/dm3 per day, TPP
levels range from <0.05 to
15 ng/dm3 per day, DBPP
levels range from <0.05-
20 ng/dm3 per day. TnBP
levels range from <0.05-19
ng/dm3 per day. And the
less frequently detected
tri-iso-butyl phosphate
(TiBP) levels range from
<0.05 to 0.42 ng/dm3 per
day. In the ACC the TCP
levels range from <0.9 to
270 ng/dm3 per day, TPhP
levels range from <0.05 to
7.6 ng/dm3 per day, DBPP
levels range from 1.7- 970
ng/dm3 per day. TnBP
levels range from 56-
16000 ng/dm3 per day and
the TiBP levels range from
5.6 to 390 ng/dm3 per day.
In all 6 HEPA filters from
the jet engine airplanes
TCP was detected with
levels range from 1.1 to
4.3 ng/g per hour. Again
no ortho-isomer of TCP
was detected.
“after engine replacement”.
Ortho-isomers of TCP were not
detected in any of the samples
collected in this study.
Wipe sampling is in general
favors sampling of non-volatile
OPs (TCP and TPhP) whereas
ACC sampling resulted in high
recovery for all alkyl OPs.
Still, for the long term sampling,
wipe samples were preferred
over ACC samples for the non-
volatile OPs due lower LOQs
and higher extraction recovery.
There was no differences in
concentration observed between
sampling the cockpit versus the
passengers cabin.
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Study design Data set Concentrations Conclusion Reference
In this study the
presence of VOCs in
cabin air of aircraft
were studied to
identify possible
emission sources.
A receptor model
using positive matrix
factorization was
used to couple the
measured VOC
levels with
information related
VOC sources to
identify the major
VOC sources in the
aircraft cabin.
84 air samples
(Tenax-TA tubes)
were collected during
14 flight on a B737-
800, The duration of
sampling ranged from
80 to 190 minutes.
In total 19 VOCs were
detected in air samples
collected during the 14
domestic flights. All
compounds and median
and mean levels are
reported in Table A.9 of
Appendix 3. Highest levels
were found for d-limonene
(median of 31 μg/m3),
followed by decanal
(median of 24.43 μg/m3),
nonanal (18 μg/m3),
toluene (13 μg/m3) 5-
hepten-2-one, 6-methyl-
styrene (11 μg/m3) and
benzene (10 μg/m3).
29% of the total VOC emission
was attributed to service of
humans, followed by chemical
reactions (15%), fuels (13%),
materials (12%), combustion
(12%), non-fuel oil (9%),
cosmetics and perfumes (5%),
and cleaning agents (4%).
Benzene (69%) followed by
acetic acid (11%) and octanal
(10%) attributed to the total VOC
concentration related to non-fuel
oils.
Wang et al. (2014)
The materials
research laboratory
was requested to
identify the chemical
composition of the
odorous vapours in
the environmental
control system of the
Royal Australian Air
Force (RAAF)
Hercules C-130
aircraft.
Bleed air samples
were collected (using
Porapak Q adsorbent
tubes) from the bleed
air adducts from the
cargo/cabin
compartment, while
the engine was
operating in a variety
of situations. 4 aircraft
were sampled during
the fight. One of the
four was also sampled
on the ground. From
each airplane 5
samples were
collected. Additional
pyrolysis experiment
were performed with
Avtur jet fuel at 25 °C,
and with the 2
hydraulic fluids (MIL-L-
23699C, NATO 0-146,
MIL-H-5606E NATO
H515) at 100 and 200
°C. The Exxon 2380
was also pyrolysed at
450 °C.
TCP was not detected in
any of the air samples
collected.
However some trace
levels of
organophosphorus
compounds, particularly
TCP was found in the air
filter bags.
Avtur jet engine leakage
from the fuel nozzle
produces a continuous
background of
hydrocarbon vapours (0.1
-0.5 PPM).
No evidence of any vapour
contamination, other than
avtur, from in-flight air
samples was found.
Trace levels of the
neurotoxic
trimethylolpropane
phosphate (TMPP) was
detected during the
laboratory pyrolysis
experiment. However
there was no evidence
that this compound is
present in samples taken
from the aircraft.
No evidence was found that
neurotoxic bicyclophosphorus
compound derived from the oil
additive are present in the cabin
air.
The authors recommend that
additionally to the normal
maintenance, the use of
charcoal cloth filters need to be
further investigated to absorb
the noxious odours.
Kelso et al. (1988)
This study
investigated the
presence of TCP in
cabin air from two
airplanes. The focus
in this study is on
the mono-, di- and
tri-ortho-TCP.
90 air samples were
collected during 26
flight on two airplanes
which had two Rolls-
Royse turbine
engines. Samples
were taken during the
take-off (25 min) and
during the total flight
(5h) with 2 L/min.
In the engine oils (Mobil jet
oil II) the ortho-TCP levels
were < 20 μg/kg).
In 15% of the samples
o,o,o-TCP was detected
with levels ranged from 2
to 65 ng/m3. This was
during normal flight
conditions.
The total TCP
concentration in the air
samples ranged from 17 to
167 ng/m3.
Strong correlation (R2 = 0.81)
between the tri(o,o,o)-cresyl
phosphate levels in air of the
cockpit and air collected in the
passenger cabin was observed.
The total TCP concentration is
higher during take-off compared
to the samples taken over the
entire flight.
Rosenberger et al. (2013)
The aim of this study
was to develop a
procedure to monitor
TCP in cockpit and
cabin air of an
aircraft. By using this
3 different airplanes
from the ADF were
sampled: the fighter
trainer (FT), cargo
transport (CT) and
fighter bomber (BF).
Mono, di or tri(o,o,o)-
cresyl phosphate were
below LOD in all collected
samples. The following
TCP isomers (mmm,
mmp, mpp and ppp) were
The highest level observed was
51.3 ug/m3 during this flight
smoke and odour was reported.
However, the report of smoke
and odour did not necessarily
correlate with TCP
Denola et al. (2011)
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Study design Data set Concentrations Conclusion Reference
method the TCP
concentration in
Australian defence
force (ADF) aircraft
were measured in
order to assess
potential risks for the
exposure to TCP.
Long duration air
sampling was
performed with 0.06 g
of Porapak-Q glass
tube with 2 L/min.
Short term air
sampling was
performed with
metricel filters (GN;
0.8 μm) at 36 L/min.
reported in some of the
samples and were
reported as total TCP.
In only 11 of the total of 78
samples the total TCP
levels were just above the
LOQ with total TCP levels
range from 0.12 to 4.99
μg/m3. In only 2 samples
the total TCP levels (21.7
and 51.3 μg/m3) were
higher than 10 times the
LOQ.
Other peaks were present
in the gas chromatogram
with similar retention times
as TiBP, TnBP and TPhP.
concentrations in other
incidents. In two flights smoke
was reported however no TCP
was detected above the LOQ.
The results of this study indicate
a low health risk from TCP
exposure.
This study
investigated the
health complaints by
flight crew as well as
the finding regarding
air quality
measurements
taken during test
flight conditions on
two BAe 146 aircraft
that experienced oil
seal failures.
The findings were
compared with two
BAe 146 aircraft and
a Dash 8 aircraft
that never had been
associated with any
complaints.
Aircraft 1: complaints
about air that made
flight crew ill. BAe 146
was flying on Castrol
5000. In the evening
the oil was replaced
with Exxon 2380.
VOCs were sampled
using charcoal
absorbents tubes (0.1
L/min) and higher
molecular weight
hydrocarbons with
filter cassette (2
L/min).
Aircraft 2: complaints
about air quality during
2h flight. CO and CO2
measurements were
performed.
Aircraft 3: BAe 146 no
complaints monitored
for 3 h flight. Aircraft 4:
no complaints
monitored for 4 h with
charcoal filter.
Aircraft 5: monitored
for 3h.
Aircraft 1; BAe 146
sampled for 1.5 h. 2 min
after take-off oily smell.
During test flight number
of ascent and descents
were made to simulate
take-off and landing.
VOCs detected in the air
were long chain
hydrocarbon derivatives,
3,7-dimethy-1,3,6
octatriene, 3-isopropoxy-
1,1,1,7,7,7 hexamethyl-3,5
and 5 siloxane derivatives.
No clear differences
between VOC detected in
the cockpit compared to
the rear end of the aircraft,
with the exception of
hexadecamethyl
heptasiloxane which was
only found in the rear end
of the aircraft. The sources
of these compounds were
not identified in this study.
Major oil compounds could
not be detected in the air
of aircraft 1. They may be
filtered out by the APU or
condensed in the
ventilation system.
In all airplanes the CO2
levels increased after
landing of the passengers
and before take-off, and
decreased until landing. In
aircraft 2 the CO2 levels
ranged from 528 to 900
ppm. The CO2 levels in
aircraft 3, 4 and 5 ranged
from 800-2300 ppm. CO
was not detected in any of
the airplanes above the
LOD of 1 ppm. O2 levels
decreased after take-off
and increased prior to
landing. The average O2
percentage in the
airplanes was 21.7 %.
TCP was not detected in the air
of the aircraft that suffer from
smoke odour and oil leakage.
In flight oil seal failure in jet
engines of BAe 146 aircraft was
traced as the source of smoke in
the cabin.
Van Netten (1998)
Summary of aircraft Symptoms were In this study no The symptoms reported by Van Netten (2005)
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Study design Data set Concentrations Conclusion Reference
air quality incident,
symptoms,
exposures and
possible solutions.
reported by aircrew
members flying for an
unreported airline
company on a MD-80
aircraft and by aircrew
from another
unreported airline
company flying on a
mixed fleet of aircraft.
measurements were
performed on oil
compounds in air planes.
aircrew members appear very
consistent with the symptoms of
CO and TCPs. Exposure
measurement in airplanes during
air quality incident is rare but is
needed to connect the
symptoms observed by aircrew
members to those that have
been reported for these agents
in the literature.
Investigation of the
presence and
concentration of five
TCP isomers (ooo-
TCP, mmm-TCP,
mmp-TCP, mpp-
TCP and ppp-TCP)
in the cockpit of 737
Boeing aircraft
under normal flight
conditions and
during the operation
of the auxiliary
power unit (APU)
only.
80 air samples were
collected from 4 B737-
700s, 3 B737-800s, 3
B737-900s and from 2
B737-700s and 800
while running the APU
on the ramp. Sampling
was performed using
Chromosorb 106 in
glass tubes in
combination with glass
filters. Wipe samples
were taken before and
after the flight.
No events were reported
during any of the flight.
In 37 of the 80 air samples
measureable TCP-isomers
have been detected. The
levels ranged from (the
lowest detectible level) 0.5
ng/m3 to 155 ng/m
3, with
an average of 6.9 ng/m3.
Highest TCP levels were
observed during climb and
descent. The TCP levels
observed in the wipes
ranged from 0.01 to 0.06
ng/cm2.
According to the authors, it is
likely that the emission of
particles containing TCP
isomers in the cockpit is
discontinuous.
No detectible ooo-TCP was
found in the air samples, wipe
samples or oil samples analysed
in this study.
TNO report (2013b)
Analysis of cabin air
for VOCs, SVOCs,
particles and CO
under normal flight
conditions and
during fume or air
quality events.
100 flights in 5
different aircraft (B757
cargo, B757, A320/1,
BAe 146 and A319)
were monitored in this
study. Sorbent tubes
containing Tenax TA
were used for the
sampling. Besides
total VOCs and
ultrafine particles
numbers the following
target compounds
were included in this
study: ooo-TCP, the
other TCP isomers,
TnBP, toluene,
meta+para-xylenes,
limonene,
tetrachloroethylene
(TCE) and undecane.
Concentrations of toluene,
limonene, xylenes,
undecane and TCE in
cabin air were comparable
with levels observed in
homes in developed
countries.
Total VOCs levels were
mostly below 2 ppm.
Higher levels were
reported during air quality
events.
Levels of CO are in some
cases even higher in
homes than in the aircraft
cabin and were mostly
below 2 ppm.
In more than 95% of cabin
air samples no total TCP
of ooo-TCP was detected.
Highest ooo-TCP level of
22.8 μg/m3 was observed
during climb of the aircraft
(overall mean 0.07 μg/m3).
The overall mean total
TCP levels was 0.14
μg/m3 (with a maximum of
28.5 μg/m3). The highest
TnBP levels recorded was
21.8 μg/m3 with an overall
mean of 1.07 μg/m3.
No fume events occurred that
triggered the airline’s protocols
for formal reporting of incidents.
However during 38 flights,
fumes/smell events were
reported in the post flight
questionnaires. Samples
collected during air quality event
did not contain elevated levels of
any of the target compounds
included in this study.
Crump et al. (2011), part 1
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Analysis subcategory 2: In vitro or in vivo toxicity tests conducted with aviation engine oil or its 3.4.3
fumes
Temperature ranges for pyrolysis experiments on jet engine oils.
The pyrolysis experiments described in the selected papers were performed at various temperature ranges.
Examples are summarized below.
Crane et al. (1983) used temperatures ranging from 300 to 600°C for the evaluation of thermal degradation
products from aircraft engine oil temperatures ranging from 300 to 600°C. No specific information was given on the
selection of the temperature range, only that measured carbon monoxide (CO) occurred at 306°C and doubled in
concentration when the temperature increased from 350 to 533°C (up to 10600 ppm). Crane et al. (1983)
concluded that the temperature of 400°C is an adequate model for thermal degradation in the turboprop engine.
Crane et al. (1983) also concluded that none of the formed pyrolysis products generated was more toxic to rats
than the quantity of CO that was formed.
Porvaznik et al. (1987) evaluated the acute dermal toxicity of a thermally decomposed military specification
synthetic aircraft lubricant. The temperature of the pyrolysis experiment ranged from 300 to 700°C. Again no
specific information was given for temperature range used in this paper. However, the formation of
trimethylolpropane phosphate (TMPP) was evaluated showing that it was formed at temperature around 440°C,
and increased significantly in concentration with temperature. Provaznik et al. (1987) concluded that TMPP was
formed during pyrolysis of the engine oil if the temperature was higher than 400°C. The engine oil contained
trimethyl propane triheptanoate (TMP) and tricresyl phosphate (TCP) or triaryl phosphate (TAP).
Van Netten et al. (2000, 2001) and van Netten and Leung (2000) used a temperature of 525°C to perform pyrolysis
experiments on various engine oils. Bleed air was diverted from a location just prior to the engine combustion
chamber at a temperature around 500°C. Van Netten et al. (2000, 2001) and van Netten and Leung (2000) also
mentioned that 525°C is the optimum temperature for the formation of TMPP.
The overview of Chaturvedi et al. (2010) specified that oil leaks related to dysfunctional seals can be subjected to
temperatures of 500°C and higher.
Hildre and Jensen (2015) observed that bleed air from the high compression stage ranges from 350 to 600°C,
whereas it ranges from 100 to 300°C when taken from the low stage compressor (Spittle, 2003).
Michaelis (2011) reports that fogs formed during pyrolysis of the jet engine oil (MIL-L-7808) at temperature ranged
from 204 to 288°C were less toxic than those formed at 315°C (Treon et al., 1954, Treon et al., 1955).
Michaelis (2011) reports that typically high-stage engine compressor temperatures can vary from 300 to 650°C
(Spittle, 2003) or from 450-600°C (AT&H. 2000) and go up to 650°C in a B767 at take-off power (Hunt et al., 1995).
In the report of National Research Council (2002) much lower temperatures were reported. Those do not exceed
temperatures higher than 350°C (Table 3.7).
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Table 3.7. Typical conditions from bleed air of an aircraft engine (Table is copied from National Research Council
(2002)).
Overall, it can be concluded that the temperature in an aircraft engine compartment, where oil vapour and pyrolysis
products may be formed, can reach temperatures above 500°C. It was also observed that more toxic fumes
(containing CO and TMPP) were generated at temperature higher than 400°C.
The analysis of subcategory 4: Composition of aviation engine oil, fumes or pyrolysis products. 3.4.4
Composition of the aviation engine oils.
Porvaznik et al. (1987) reports that military specification L-23699 synthetic aircraft lubricants contains trimethyl
propane phosphate (TMP), pentaerythritol monobutyrate triheptanoate (PE), tricresyl phosphate (TCP), or triaryl
phosphate (TAP).
Van Netten (1999) analyzed jet engine lubricant oils for toxic element. However no toxic elements (such as lead,
mercury and thallium) were identified in any of the engine oils (Exxon 2380, Mobil and Castrol 5000).
Van Netten et al. (2000, 2001) and van Netten and Leung (2000) measured the composition of two commercially
available jet engine oils (Castrol 5000 and Exxon 2380). Various compounds were detected in the oil samples
which are listed in Table 3.8. It is noted that the identified compounds in Table 3.9 were not all confirmed with
appropriate standards.
Winder and Balouet (2002a) examined the ingredients in jet engine oils and indicated that at least two ingredients
are hazardous: N-phenyl-1-naphthylamine and tricresyl phosphate (TCP). Other compounds listed on the material
safety data bulletin (MSDB) from Mobil jet oil are listed below.
• Synthetic esters (mixture of 95% C5 – C10 fatty esters of pentaerythritol and dipentaerythritol.
• 3% tricresyl phosphate
• 1% phenyl-α-naphthylamine (N-phenyl-1-naphthylamine) (PAN) (CAS No. 90-30-2)
• Benzamine (4-octyl-N-(4-octylphenyl) CAS No. 101-67-7)
Winder and Balouet (2002a) report that the commercial product of N-phenyl-1-napthylamine is 99% pure, however,
can contain the following six impurities N-phenyl-2 naphthylamine (500 to 5000 ppm), 1-naphthylamine (below 100-
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500 ppm), 2-naphthylamine (below 3-50 ppm), aniline (below 100-2500 ppm), 1naphthol (below 5000 ppm) and 1,1
dinaphthylamine (below 1000 ppm).
De Nola et al. (2008) screened jet engine oils for the presence of 10 isomers of TCP. De Nola et al. (2008) were
able to separate 9 of the 10 isomers by GC-MS/MS (only the omm-TCP and oop-TCP were coeluting). The ortho
isomers of TCP observed in the jet engine oils consist almost exclusively of mono-ortho-isomers in a concentration
range of 13-150 mg/L.
The mmp-, mmm- and mpp- TCP isomers were dominating in the jet engine oil. The omm- and omp-TCP isomers
were found at low concentrations while the opp-, oox- and ooo-TCP isomers were not detected. The ratio of the
m/z observed for the isomers are different. This helps identifying and separating the isomers. Whereby m/z 165
fragment ion was the base peak for all the six ortho-isomers. m/z 168 was the base peak for the four isomers
containing only p- and m- substituents. An m/z 243 fragment was also characteristic of the xxx-isomers (where x=
m or p). Also m/z 277 was more abundant in the o-isomers compared to the m- and p- isomers. However, the
intensity may vary with instrument operating conditions.
Other compounds that have not been mentioned above, which were summarized in the following review report
(Expert Panel on Aircraft Air Quality, 2011) are listed below.
On the MSD of Mobil jet oil II from Australia and Canada is written that it contains <2% alkylated diphenyl amines.
NYCO SA produces Turbonyciol 600, which contains triphenylphosphate (TPhP) rather than TCP.
OHRCA (2014) describes the analysis of four isomers of TCP (ooo-TCP, mmp- TCP, mpp-TCP and ppp-TCP) in
nine jet engine oils. In three of the nine engine oils (Aeroshell 560, BP 2389 and BP2197), ooo-TCP was detected
with a concentration of 0.01%, just at the detection limit. Overall the TCP isomer patterns observed in the nine
engine oils were comparable, with mmp-TCP as the most dominant isomer followed by mmm-TCP, mpp-TCP and
ppp-TCP with an relative concentration of 49%, 29%, 22% and 0.2%, respectively. In six of the nine jet engine oils
(Mobil II,BP 2380, used BP2380, Mobil 291, Exxon O-156 and Mobil 245) the total-TCP concentration was around
5%, which is higher than the 3%, which is often referred to in the MSDS for these oils. OHRCA (2014)(OHRCA,
2014) also reports that in NYCO jet engine oil, TCP was replaced by triisopropyl phenyl phosphate (TIPP).
Whereas others have reported that in the NYCO jet engine oils, TCP is replaced by TPhP.
The compounds observed in jet engine oils and hydraulic fluids by Hildre and Jensen (2015) are listed below.
• N-phenyl-1-naphthylamine (CAS No. 90-30-2)
• Alkylated diphenylamine (CAS No. 122-39-2)
• Butyl diphenyl phosphate (CAS No. 2752-95-6)
• Dibutyl phenyl phosphate (CAS No. 2528-36-1)
• Isopropylated triphenyl phosphate (CAS No. 68937-41-7)
• Phenol, dimethyl-, phosphate (3:1) (CAS No. 25155-23-1)
• Tributyl phosphate (CAS No. 126-73-8)
• Tricresyl phosphate (CAS No. 1330-78-5)
Note that the compounds listed above could be present in jet engine oils or in hydraulic fluids. No distinction was
made in this report.
Pyrolysis and thermal degradation products formed during pyrolysis of aviation engine oils.
Crane et al. (1983) concluded that during pyrolysis of six jet engine oils (including Exxon 2380, 2389 and Mobil II
jet oil) none of the products generated a smoke component that is significantly more toxic to rats than the quantity
of CO produced. CO was the only pyrolysis product identified in this paper.
Provaznik et al. (1987) only quantified TMPP in the condensate of the pyrolysis engine oils at temperatures higher
than 440°C.
Van Netten et al. (2000, 2001) and van Netten and Leung (2000) performed pyrolysis experiments on two
commercially available jet engine oils (Castrol 5000 and Exxon 2380). The pyrolysis experiments were performed
for 1 min at a temperature of 525°C. Various pyrolysis products were identified and are listed in Table 3.9. It is
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noted that the identified compound in Table 3.9 were not all confirmed with appropriate standards). No evidence
was found for the generation of NO2 and HCN but the CO2 and CO levels increased in time. However, TMPP was
not detected in this study. Some of the volatilized and pyrolysis produces generated in this study may not reach the
cabin air because they may condensate onto the ducts of the aircraft ventilation system. This indicates that through
condensation some of the pyrolysis products accumulated onto the ducts of the ventilation system. This was also
observed by Rubey et al. (1996) for TMPP, which was not observed it the gaseous phase during incineration of jet
engine oil but in the scrapings of the boiler walls.
Therefore, it would be unlikely that TMPP produced during pyrolysis would end up in cabin air, unless the
temperature of the ducts for some reason would become elevated.
Winder and Balouet (2002b) examined the ingredients of jet engine oils and suggested that the following chemical may release after pyrolysis of jet engine oil;
• Combustion gases such as carbon dioxide and carbon monoxide • Other irritating gasses, such as oxides of nitrogen • Partially burnt hydrocarbons (including irritating and toxic by products, such as acreolein and other
aldehydes) • TCP and TCP thermal degradation products (TCP boils at 420°C)
Ramsden et al. (2013) suggested that the higher ortho-TCP/TCP ration observed in bleed air compared to the
ration observed in the engine oil may be related to isomerization of the TCP within the engine during operation.
Investigation on the isomerization of cresol at 380°C using a solid phase catalyst resulted in an equilibrium
composition of 36% ortho, 48% meta and 16% para.
The National Research Council (2002) reports that formaldehyde, acetaldehyde and acreolein could be found in
engine oil or could be formed during thermal decomposition of engine oil (Nagda et al., 2001).
CAA (2004) reports that jet engine oil breakdown products could contain over 40 different chemicals, and that most
of them have no published toxicity data. Various organic acids were observed in the pyrolysed jet engine oils. Most
likely the short-chain organic acids such as valeric and pentatonic acid may be responsible for the “old sock” odor
observed in airplanes. Furthermore, differences in composition were noticed between used and new oil both for
unpyrolysed oil as well as for pyrolysed oil (at 350°C; 350°C high humidity and 450°C). It also analysed the various
compounds observed in the environmental control system ducts from BA 146 aircraft. Figures 3.1-3.4 show the
results.
Overall, TCP isomers and TMPP were the most frequently reported compounds in the engine oil or formed during
pyrolysis. It is obvious that in most earlier studies, TCP isomers and TMPP were the compounds of high interest.
However, many other compounds are present in the oil and even more can be formed during pyrolysis of the jet
engine oil. The compounds listed above and listed in Table 3.8 and 3.9 can be used to generate a suspect
compound list to screen the engine oils tested in this study. During pyrolysis many compounds may be formed. The
resulting products formed are highly dependent on the conditions under which the pyrolysis experiment is
achieved. Besides the TCP-containing turbine engine oils, nowadays TCP-free engine oils are available that
contain TPhP or TIPP instead of TCP. It would be interesting for the pyrolysis experiment performed in our study to
see whether the pyrolysis product profile would be different between TCP-free and TCP-containing turbine engine
oils.
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Table 3.8. Various compounds detected with GC-MS in two jet engine oils, Castrol 5000 and Exxon 2380. (Table is
copied from Van Netten and Leung (2000).
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Table 3.9: Various compounds detected with GC-MS in two pyrolysed jet engine oils, Castrol 5000 and Exxon 2380. (Table is copied from Van Netten and Leung (2000).
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Figure 3.1. Pentaerythritol (PE) esters identified by GC-MS in pyrolysis products of new and used oils. Details of
methods of pyrolysis and analysis are given in Marshman (2001) (Data used is the property of BAE Systems)
(Figure is copied from CAA (2004)).
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Figure 3.2. Trimethylpropane (TMP) esters identified by GC-MS in pyrolysis products of new and used oils. Details
of methods of pyrolysis and analysis are given in Marshman (2001) (Data used is the property of BAE Systems)
(Figure is copied from CAA (2004)).
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Figure 3.3. Tricresyl phosphate (TCP) esters identified by GC-MS in pyrolysis products of new and used oils. Details of methods of pyrolysis and analysis are given in Marshman (2001) (Data used is the property of BAE Systems) (Figure is copied from CAA (2004)).
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Figure 3.4. Organic acid, ketone and amine contaminants identified by GC-MS in pyrolysis products of new and used oils. Details of methods of pyrolysis and analysis are given in Marshman (2001) (Data used is the property of BAE Systems) (Figure is copied from CAA (2004)).
Selection of oils 3.4.5
In the tender a proposal was made by EASA for selection of several (at least two) aviation turbine engine/APU oil
brands present on the market, in order to purchase and use them in the frame of this study. The selection shall
consider oil brands which are widespread among commercially operated large transport airplanes.
In close consultation between the consortium and EASA, a selection was made of two most commonly used
brands of oils for different aircraft types covering the European main fleet.
The selection contains the following candidate oils:
Unused engine oil typically for category twin-aisle/ long range (B747/A340)
Unused engine oil typically for category single aisle/short range (B737/A320)
Used engine oil typically for category single aisle/short range (B737/A320)
KLM provided the consortium the above mentioned engine oils. For each oil brand three one litre new oil cans of
same batches were delivered to TNO. KLM provided the used engine oil in six different cans, which were mixed
and homogenized by TNO and put into one batch.
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4 Task 2: Chemical characterisation of jet oil
4.1 Introduction
Pyrolysis is a thermochemical decomposition of organic material at elevated temperatures in the absence of
oxygen. It may involve the simultaneous change of chemical composition and physical properties and is
irreversible.
It is a fact, that simulations in a laboratory environment are not a 1 to 1 representation of the exact engine
circumstances that occur during real flight conditions. For example, important combustion gases like nitrogen
oxides, emitting from engines, and ozone, which is a constituent of ambient air, may cause reactions with
uncontrolled emissions of oil. For the AVOIL study the reactions with ozone and nitrogen oxides were not included
in the experiments due to the absence of sufficient valid data of typical concentration ranges for these gases in the
troposphere and in between ground level and cruise height. For ozone counts that concentrations in the
troposphere vary enormous for different tropospheric flight areas and therefore it would cost a lot of time to get well
verified data on these concentrations. Thus for both ozone and nitrogen oxides, it is difficult to define a certain
concentration in order to make a proper design for experimental conditions. In advance, we recommend therefore
to study the chemical interactions between nitrogen oxides and ozone with possible oil emissions in a separate
research project.
4.2 Design of a test methodology for the chemical characterization of oil vapours
Design of a simulation test set-up 4.2.1
A simulation for bleed air was developed including a sampling system. Within the developed simulation system the
temperature and all in and out going flows are adjusted and controlled.
The simulation system is schematically shown in Figure 4.1. How it was build is presented in Figure 4.2 ‘the
laboratory simulation system’. The different parts are described in details in the coming paragraphs.
Figure 4.1. Experimental set-up of the simulation system. Purified air is led over the heated jet oil in the reaction
chamber. Additionally a stream of purified air is led into the emission chamber to ensure that the overall flow out is
similar to the flow in. The air is mixed in the emission chamber, causing a dilution compared to that of the indoor air
in cabins. Here also a dedicated system takes care for air circulation. Contaminated air is pumped out of the
emission chamber and is led through a sampling system.
EmissionChamber
ReactionChamber
Flowpurified air
Flow Purified air
Flow Oil vapor
Air circulation
Contaminated Air out
Flow exhaust
Fresh oil supply
SamplingSystem
Volatiles
semi volatiles
particle numbers
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Figure 4.2. The laboratory simulation set-up. The following elements can be seen:
1. Reaction chamber (programmable temperature controlled oven)
2. Stainless steel emission chamber and recirculation pumps
3. Stainless steel sampling devices for (semi)volatile compounds
4. Particle number monitor
5. Glass manifold sampling device for volatile compounds
6. Carbon monoxide monitor
7. Automated syringe pump device
Reaction chamber 4.2.2
The reaction chamber (Figure 4.2; no. 1), consists of a temperature controlled (programmable) oven in which the
glass reaction vessel with the oil is placed. The reaction vessel consists of a three stainless steel channel system
(see Figure 4.3).
1) The input channel is for adding fresh purified air into the vessel to create a transport flow of oil vapour.
2) The output channel is for streaming the oil vapour to the emission chamber.
3) The supply channel is used to supply fresh oil into the reaction channel. For this purpose an automated
syringe device was directly coupled to this channel.
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Figure 4.3. The reaction vessel with the three channels for the supply of fresh oil, supply of purified air and to
stream the contaminated air out of the vessel to the emission chamber.
Emission chamber 4.2.3
The second section is the emission chamber which simulates the cabin air.
The simulation of a cabin air indoor environment is not easy as the cabin air is not only dependent of the type of
aircraft and its use of ventilation system but also on other parameters e.g. number of passengers. In general a
cabin can be seen as a box, where also air will enter, circulate and leave the cabin.
Samples of the diluted oil vapours were directly taken from the emission chamber.
This chamber has the possibility for ventilation and recirculation (including setting the air velocity over the surface).
The emission chamber is made of stainless steel, its dimensions are 43 * 45 * 52 = 100,620 cm3 = 0.1 m
3.
This chamber gives the following possibilities:
To reach quickly a homogeneous mixture of the polluted air stream.
To dilute high concentrations of oil vapour
To sample under room temperature conditions under inert conditions
To use the chamber as a functionality for collecting oil condensation
To apply easily different ventilation rates and recirculation.
Flexibility in inputs and outputs for sampling devices.
Figure 4.4 shows the inside of the stainless steel emission chamber and the different flow connections on top and
on the right side of the chamber. On the top one connection where the oil vapour enters the chamber and on the
right side 5 connections for establishing sample points and flow in/outputs.
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Figure 4.4. The stainless steel emission chamber and its connectors for in- and output of flows.
The sampling system 4.2.4
Two important boundaries for sampling have been defined and therefore a novel dedicated sampling system was
developed. The defined boundaries were:
Sampling should not affect the total flow including air speed in the emission chamber.
If there is no sampling, the flow should behave as a bypass, and thus not affecting the total flow including the
air speed in the emission chamber.
Leak tests
Leak tests were carried out before each test. In order to check certain increase or decrease in flow during the tests,
the total flow out of the simulation system was measured continuously.
All flows of the individual sampling devices were controlled by mass flow units and before start and end of each
simulation test, flows were measured with a calibrated flow device.
Preventing losses caused by adsorption
In order to prevent losses caused by adsorption to surfaces of semi volatiles and for compounds with relative low
vapour pressure, the inlet of the sampling devices for polycyclic aromatic hydrocarbons (PAHs), mineral oil and
organophosphate esters (OPEs) were directly connected to the emission chamber. The inlet of the particle number
monitor was positioned in the middle of the emission chamber. This is shown in Figure 4.5 ‘the sampling devices
for PAHs, mineral oil and OPEs’.
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Figure 4.5. The sampling devices for PAHs, mineral oil and OPEs, coupled directly to the emission chamber.
The glass manifold
The volatile compounds like VOCs2, aldehydes and carbon monoxide were directly measured from the glass
manifold which is connected via a short Teflon tube to the emission chamber (see Figure 4.6).
Figure 4.6.The sample points connected to the glass manifold. Carbon monoxide was sampled via the blue line
(left), VOCs were sampled using two TenaxTM
GR tubes (middle), and aldehydes were sampled via 2,4-
dinitrophenylhydrazine (DNPH) cartridges (right).
Advanced observation
In advance, the following observation have been made. During the oil simulation tests some condensation of oil
took place (see Figure 4.7) near by the oil vapour outlet of the emission chamber and at the front of the glass
manifold.
2 VOCs are defined as organic volatiles, which boil between 50 and 260°C.
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Figure 4.7. Left the condensation of oil nearby the vapour inlet can be seen, while on the right condensation of the
oil inside of the emission chamber after a simulation run can be seen.
In order to minimize losses of semi volatiles (compounds with relative low vapour pressure) during sampling, the
inlet of the sampling devices for polycyclic aromatic hydrocarbons (PAHs), mineral oil and organophosphate esters
(OPEs) were positioned directly in the emission chamber. Due to condensation of oil in the chamber,
concentrations of PAH, mineral oil and OPE’s might be slightly underestimated.
Realising having leakages of oil under real flight conditions, the same physiological effect will take place as in the
simulation experiment.
In case of real oil leakage, evaporation of the oil takes place due to high temperatures in the compression
chamber, and shall condensate because of temperature drops downstream of the compression chamber towards
the packs and cabin. Condensation of oil in the simulation test will gave some losses of semi volatile compounds. It
must be emphasized that the reported measured concentrations of semi volatiles are therefore underestimated
results due to condensation. As already mentioned before, condensation of oil will also be the case in real flights
when leakage of oil takes place.
4.3 Performance of the simulation tests
Experimental 4.3.1
The simulation tests were carried out using the equipment and analytical procedures as described in section 4.2. In
summary a reaction vessel with the oil was placed in the oven. A flush of purified air was applied to transport the oil
vapour produced at the given temperature. This flush of contaminants was mixed in the emission chamber with
purified air. As the door of the emission chamber is provided with a rubber seal no contaminants can leave the
emission chamber other than by the sampling system. The contaminated air was led through the different samplers
and analysed afterwards. Particle number concentrations, carbon monoxide and temperature were continuously
monitored during the whole simulation tests. In between the oil experiments blank system (without oil) experiments
were carried out before each new simulation test.
The simulation set-up was placed in a laboratory environment, the temperature and relative humidity were adjusted
by the climate system present in the lab (approx. 20 to 22°C and 55% relative humidity). All tests were conducted
under normal standard atmosphere.
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Temperature conditions during simulation tests 4.3.2
From the literature it is observed that pyrolysis experiments were performed at various temperatures in the range
from 200 to 600°C. It is clear that bleed air temperature depends on air taken from high or low compression stages
and type of engine. Although various laboratory studies are performed with respect to pyrolysis, it is not always well
defined and supported by real measurements what the temperature profile is for typical normal bleed air
temperatures. In the report of National Research Council (NRC) (2002) flight profiles were made for typical
conditions from bleed air of an aircraft engine which do not exceed temperatures higher than 350°C. The
information in the NRC report on the temperatures are referred to as ‘typical conditions of bleed air from engine'.
The provided source is ‘Responses from Boeing to committee questions, July 13, 2001’. No further information on
the type of engine or aircraft is provided.
ADSE Consulting and Engineering, who acts as an independent expert, to assist in case of operational questions
for the consortium, was asked to work out bleed air characteristics for pressure and temperature. A flight profile
and aircraft (100 to150 seats) was chosen for a typical European flight of 500 nautical mile (NM). A typical current
generation engine was taken. The results of the bleed air pressure and temperature are listed in Table 4.1.
Table 4.1. Bleed air pressure and temperature for a typical European flight
Movements Bleed pressure Bleed temperature
kPa absolute (°C)
Taxi at Ground idle 150 110
Max take off at 0 ft 940 350
Max climb rating at 1,500 ft 880 350
Max climb rating at 37,000 ft 340 300
Cruise 310 270
Flight idle at 37,000 ft 90 130
Flight idle at 1,500 ft 160 120
Holding at 18,000 ft 350 240
Ref: Vliegtuigomstandigheden ten behoeve van EASA test, 16-ME-013 V1.0, 11 February 2016, (ADSE)
The outcome of the ADSE investigation was discussed during the interim meeting in February 2016 at EASA. It
was decided to take 350°C as a valid maximum temperature for the AVOIL study. Furthermore these numbers
were checked at the KLM-Engineering department and were confirmed to be representative for typical worst-case
temperature/pressure circumstances.
For the chemical characterization of the simulation tests the following conditions and settings profile were applied
as described in Table 4.2:
Table 4.2. Conditions and settings for the chemical characterization of the simulation
and blank tests.
Simulation test Samples taken during
simulation test
Sampling time
(min)
Oxygen content
(%)
Temperature
(˚C)
Oil supply during
simulation test
Ground level to top of climb sample 1 30 20.9 20-350 no Cruise sample 2 60 20.9 350 yes
All tests are conducted under normal standard atmosphere.
During the first part of the simulation tests volatile compounds will be released from the oil during the warming-up
of the oil from 20 to 350°C resulting in exhaustion of the oil before the second part (Cruise) of the sampling starts.
Therefore oil is only continuously dropwise introduced in the reaction vessel during the cruise sampling period of 60
minutes at 375 (± 25)°C.
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Experimental settings of the simulation tests 4.3.3
For all simulation tests air flows for the different samplers, temperature range and time were set. Tables 4.3 and
4.4 show the settings of air flows and temperature during the simulation tests. In order to avoid under- or
overloading of analytes, air flows were chosen based on the applicability of the individual sampling methods.
Table 4.3. Air flow settings of the simulation system
Flow settings
simulation tests
Controlled
values
Flow purified air through reaction
vessel (=oil vapour entering the
cabin)
380 ml/min
Sampling flow mineral oil 2000 ml/min
Sampling flow OPEs 2000 ml/min
Sampling flow PAHs 2000 ml/min
Sampling flow VOCs 42 ml/min
Sampling flow aldehydes 400 ml/min
Sampling flow Condensed
Particle counter 300 ml/min
Sampling flow CO-monitor 1100 ml/min
Sampling flow Bypass 3000 ml/min
Total flow out 10842 ml/min
Ventilation rate 6.5 h-1
Table 4.4. Temperature settings during the simulation tests
Temperature settings
simulation tests
Controlled
values
Temperature laboratory (20 ± 2)°C
Relative humidity laboratory (60 ± 5)%
Temperature Emission chamber
during simulation test (21 ± 1)°C
Temperature oil during simulation
test 0-30 min (21 to 350)°C
Temperature oil during simulation
test 30-90 min (375 ± 25)°C
Selected oils for the simulation tests 4.3.4
For the simulation tests the following engine oils were tested (see also Table 4.5):
Oil An: new oil of brand A
Oil Au: used oil of brand A. History: was taken from a certain Boeing 747 Freighter. Hours since last shop visit
13653, Cycles since last shop visit 2615.
Oil Bn: new oil of brand B
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Table 4.5. The properties of selected oils
Property3 Oil An Oil Bn
Date Safety Data Sheet March 30, 2015
march 17, 2015
Pour Point (˚C), ASTM D 97 -54 -59
Flash Point (˚C), ASTM D 92 246 > 246
Fire Point (˚C) n.a. n.a.
Autogenious Ignition Temp (˚C) n.a. n.a.
Density @15˚C, kg/l, ASTM D 4052 0.997 1
TCP (percent by weight) 1- < 2.5 1- < 3
n.a: data not available
Applied sampling and analytical methods 4.3.5
Organophosphate esters
Organophosphate esters (OPEs) were sampled using Teflon/glass fiber filters in combination with Chromosorb®
106 adsorption tubes. Extraction of the samples was carried out using accelerant solvent extraction (ASE). The
extraction solvent is a mixture of dichloromethane (DCM) and n-hexane (50:50 v/v). Before extraction an internal
standard TPh-d15 was added to the sample. After extraction the extract was concentrated followed by a clean-up
using 3% deactivated florisil. Prior to analysis an injection standard 1,2,3,4-tetrachloro naphthalene (TCN) was
added to the extract. The analyses were performed with GC-MS. The method is developed and validated by TNO
according to NEN 7777 (NEN-EN 7777:2011) and described in an internal TNO report (TNO, 2013a).
Mineral oils
Mineral oils were sampled using glass fibre filters in combination with XAD-2 adsorption tubes. Extraction of the
samples was carried out using hexane. After the extraction, the extract was pre-concentrated followed by a clean-
up with florisil. The final extract was analysed with Gas chromatography-Flame Ionisation Detector (GC-FID) . Both
the identification and quantification of the compounds are based on an external standard. The sampling method is
based on NIOSH 5026 (NIOSH, 1996), the analysis method is based on NEN 6978 (NEN 6978:2016).
Polycyclic Aromatic Hydrocarbons (PAHs)
PAHs were sampled with a glass fibre filter in combination with XAD-2 adsorption tubes. Extraction of the samples
was carried out using toluene. After extraction with Accelerent Solvent Extraction (ASE) the extract was pre-
concentrated followed by clean up with silica gel. The final extract was analysed with GC-MS isotope dilution based
on ISO 12884 (ISO 12884:2000).
Aldehydes
Aldehydes were sampled with 2,4-dinitrophenylhydrazone (DNPH) Sep-Pak Xposure cartridges. The aldehydes in
the air reacts with DNPH and H+
to the corresponding hydrazone derivatives. Extraction of the samples is carried
out with acetonitrile.
Using liquid chromatography coupled with mass spectroscopy the determination of the compounds, quantitatively
and qualitatively, was established. Identification of the individual carbonyls in the extracts is carried out on basis of
the retention times and quantification is based on an external calibrated standard. The method is based on ISO
16000-3 (ISO 16000-3:2011).
Volatile organic compounds (VOCs)
TenaxTM
GR adsorption tubes were applied for sampling VOCs in the range of C6-C12. Subsequently the
determination of organic volatiles was carried out according to internal procedure ORG-141 ‘Determination of
Volatile Organic Compounds Using Thermal Desorption and Gas Chromatographic Analysis’. The desorption of the
adsorption tubes is carried out my means of an automatic thermo sorption unit. The desorbed compounds are
3 The Oils are used under code. The Safety Data Sheets are present at the authors office.
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trapped my means of a cold-trap and are being analysed on-line using a gas chromatograph equipped with a
capillary column which is coupled with a mass spectrometer. The identification of the compounds is based on the
retention time and mass spectrum. Quantification is performed with the use of an external standard. The method is
based on ISO 16000-6 (ISO 16000-6:2011).
Condensed particle counter (CPC) and carbon monoxide
Particle numbers are measured with the use of a butanol driven CPC 3775 (TSI) with a cut-off of 4 nm to 1000 nm
particle diameter. Data points were measured each second. Concentrations of particles were calculated in particle
numbers per cm3.
Carbon monoxide is measured with a Thermo monitor type 48i. measurements are based on the principle that CO
absorbs infrared radiation at a wavelength of 4.6 microns. Because infrared absorption is a nonlinear measurement
technique, it is necessary for the instrument electronics to transform the basic analyser signal into a linear output.
The 48i uses an exact calibration curve to accurately linearize the instrument output over any range up to a
concentration of 10,000 ppm.
4.4 Results and discussion
In total, six simulation runs were performed: three were carried out for blank system measurements and three were
carried out using the selected oils.
With the exception of carbon monoxide and particle number measurements, all results of the oil simulation tests
were corrected for the blank concentration levels found for the blank system measurements. The results of the oil
simulation tests are presented in this section.
Temperature and loss of weight during the simulation tests 4.4.1
It is obviously that due to the increase of temperature and the flow of purified air, losses in the total mass of the oil,
present in the reaction vessel, take place. This is happening even though fresh oil was supplied to the vessel. In
order to calculate the loss of weight during the simulation test, the mass of oil was gravimetrically determined
before and after each simulation test. As the change of mass is dependent of the temperature of the reaction
chamber, and thus the temperature of the oil, the temperature gradient was observed throughout each experiment.
The temperature of the simulation tests for oil An and oil Au are shown in Figure 4.8..
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Figure 4.8. Temperature profile simulation test for new oil An (upper graph) and used oil Au (lower graph). At the
start of the simulation test the temperature of the oils amounts 21˚C. Within 30 minutes the temperature of the oils
reached a temperature of 350˚C. During the next 60 minutes of the simulation test the temperature of the oils
amounts (375 ± 25)˚C.
The temperature of the simulation test for oil Bn is shown in Figure 4.9. It has to be stated, that this temperature
profile is equal to the profiles applied for the other oils. Table 4.6 shows the loss of weight during the test for oil An,
Au and Bn.
Figure 4.9 Temperature profile simulation test for oil Bn.
Temperature profile simulation test Bn
Temperature profile simulation test An
Temperature profile simulation test Au
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Table 4.6. Loss of weight of the applied oils after completion of the simulation test.
Oil type % loss of weight after
simulation test
An 68
Au 11
Bn 82
From the results it is clear that the % loss of weight for the used oil Au is significant lower than for the new oil An
and Bn at equal settings of time, temperature and oil mass applied per simulation test.
Based on this fact, we suggest that the used oil contains less volatile- and semi-volatile fractions compared to the
new oils.
OPEs in oil and oil vapour 4.4.2
The organophosphate ester group forms an important fraction of the jet oil and will also release from the applied
oils. Therefore, it is important to know the original OPE concentration in the applied oils prior to the simulation tests.
The results of the OPE analysis in the original oils and subsequently determined in the vapour per simulation test
are presented in Table 4.7. and 4.8..
Table 4.7. OPEs found in the original oils.
Oil type An Au Bn
Components g/kg g/kg g/kg
diphenyl (2-ethylhexyl)phosphate 0.038 0.043 0.030
tris(2-ethylhexyl)phosphate 0.048 0.020 0.025
tri(m, m ,m)- cresyl phosphate 2.5 2.2 4.1
tri(m, m, p)- cresyl phosphate 6.1 5.6 11
tri(m, p, p)- cresyl phosphate 5.4 5.2 9.5
tri(p, p, p)- cresyl phosphate 1.7 1.7 2.9
Σ TCP's (g/kg) 15.7 14.7 27.5
Σ TCP's (%) 1.6 1.5 2.8
Specification supplier (%) 1< 2.5 1< 2.5 1-3
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Table 4.8. OPEs found in the vapour off the different oils.
<: below detection limit
One of the first important result coming from the TCP analysis was the fact, that there was no presence of
Tri(o,o,o)-cresyl phosphate in the applied oils. Secondly it was found that the original oils contain the following
isomers: tri(m,m,m,)-, tri(m,m,p)-, tri(m,p,p) and tri(p,p,p)-cresyl phosphate.
The mass fraction of TCP, calculated from the analytical results of the oils, corresponds with the oil specifications
described in the MSDS sheets from the suppliers.
Looking at the mass fraction of the TCP isomers it was found that the mass fraction ratio of the TCP isomers in the
oil vapour was similar to the original oils4.
No tri(o,o,o)-cresyl phosphate was present in the oil vapours.
From the simulation test it was found that the oils heated at a steady state (350°C) emits more TCPs than
compared to the simulation whereby the temperature is heated up from 20 to 370°C.
Used oil Au showed low concentrations of TCPs in the emission chamber compared to new oil An. Oil An and oil Bn
gave comparable results.
The simulation test also shows a good correlation between the composition of TCP isomers found in the original oil
and in the oil vapours at different temperatures. Comparison of An and Au shows no significant differences in
composition of the four isomers. Figures 4.11 and 4.12 show the correlation of the TCP isomer composition in the
original oils and in the corresponding oil vapours.
4 The mass fraction per isomer is defined as the mass of isomer(a) present in the air, divided by the total mass of counting isomers.
Oil type An An AU AU Bn Bn
Temperature range (˚C) 20-350 350 20-350 350 20-350 350
Test duration (min) 30 60 30 60 30 60
Component mg/m3 mg/m3 mg/m3 mg/m3 mg/m3 mg/m3
Triethyl phosphate 0.0003 < < < < <
Tris(2-butoxyethyl)phosphate 0.028 < < < < <
Triphenyl phosphate 0.0002 < < < < <
Diphenyl (2-ethylhexyl)phosphate 0.0003 < < < < <
Cresyl diphenyl phosphate 0.002 < < < < <
Cresyl diphenyl phosphate 0.003 < < < < <
Di-cresyl phenyl phosphate 0.042 0.448 0.001 0.203 0.100 0.452
Tri (o, o, o)- cresyl phosphate < < < < < <
Di-cresyl phenyl phosphate 0.057 0.624 0.001 0.239 0.075 0.568
Di-cresyl phenyl phosphate 0.027 0.291 0.0005 0.105 < 0.259
Tri(m, m ,m)- cresyl phosphate 0.944 3.43 0.049 1.73 1.03 3.81
Tri(m, m, p)- cresyl phosphate 2.051 7.71 0.119 3.92 2.12 8.89
Tri(m, p, p)- cresyl phosphate 1.709 7.01 0.100 3.50 1.59 7.58
Tri(p, p, p)- cresyl phosphate 0.505 2.31 0.025 1.09 0.425 2.21
Σ OPE's 5.37 21.8 0.30 10.8 5.34 23.8
Σ TCP's 5.21 20.5 0.29 10.2 5.16 22.5
Σ TCP/ΣOPE's (%) 97 94 99 95 97 95
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Figure 4.11. Correlation of the TCP composition for the new oils. Left the correlation of the TCP composition in the
oil vapour versus the original oil from the new oil (An) is presented. On the right the correlation of the TCP
composition in the oil vapour versus the original oil from oil Bn is presented.
Figure 4.12. Correlation TCP composition in oil vapours for the new and used oil A. Left the correlation of the
vapours of the new and used are presented. On the right the correlation TCP composition of used oil A versus
used oil A vapour is presented.
Based on these findings we found that the composition of the TCPs, presented e.g. as mass TCP fraction, in
turbine oils (new or used) is similar to the composition of the TCPs found in the air during the simulation tests.
Based on this fact, we conclude that the four TCP isomers do not deteriorate due to the applied temperature
ranges of 20 to 350°C and at 375°C. All tests in our work were carried out in purified air, thus containing oxygen. In
previous work of Havermans and Houtzager comparable results of simulation tests were found under an oxygen
depleted atmosphere (Havermans et al., 2015).
PAHs in oil and oil vapour 4.4.3
Another novel approach for this work is dedicated to the presence of PAHs in both the applied oils and their
vapours. The main reason to include these analysis is based on the formation of PAHs during combustion of fuels,
however PAHs may be present in the original oils as well. An example of a pathway causing PAHs due to
combustion is given in Figure 4.13.
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Figure 4.13. A possible pathway of the formation of PAHs during fuel combustion, where radicals from oxidation
may play a serious role in the formation of PAHs according to Dandy (Dandy, 2007).
The results of the PAH analysis in the original oils and their vapours are presented in Table 4.9 and 4.10.
Table 4.9. PAHs present in the original oils.
<: below detection limit
Oil type An Au Bn
Components mg/kg mg/kg mg/kg
Naphthalene < 0.58 < 0.58 1.9
Anthracene 1.4 1.3 1.4
Fluoranthene 1.2 < 0.62 0.59
Total PAH's 2.6 1.3 3.8
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Table 4.10. PAHs found in the oil vapours.
<: below detection limit
Based on the results, it was found that naphthalene was present in the original oil Bn in a concentration of 1.9
mg/kg. At the other hand, the original oil An did not contain naphthalene as it could not detected above the
detection limit. However, both anthracene and fluoranthene were found in the applied oils.
By comparison the analytical results of the PAH presence in the vapour and in the original oils, it was found that the
vapour does contain more different types of PAH. Therefore, we suggest the following hypotheses:
- PAHs found in the vapour may originate from small concentrations in the original oil of which we found
concentrations below the detection limit.
- During heating of the oil, a partial oxidation takes place (partial combustion) that results in the formation of
new PAHs take took place during heating of the oil due to incomplete combustion and according to Figure
4.13.
For the used oil Au, we observed more different PAHs in its vapour than for the (liquid) oil. This implies that possible
formation of certain PAHs occurred during (incomplete) combustion and that these PAHs do not remain in the oil
but are released by its vapour.
Oil Bn contained the highest amount of PAHs, compared to the other two applied oils. The sum of PAHs found in oil
Bn were about 10 to 20 times higher compared to oil A, for both new and used one.
Mineral oil in oil and oil vapour 4.4.4
A mineral oil is defined as colourless, almost tasteless, water-insoluble liquid consisting of a mixture of
hydrocarbons obtained from petroleum by distillation. A mineral oil may contain mainly alkanes and cycloalkanes.
Alkanes are acyclic saturated hydrocarbons and cycloalkanes are cyclic structured. An alkane consists of hydrogen
and carbon atoms (Cn n=number of carbon atoms) arranged in a structure in which all carbon-carbon bonds are
single. For each simulation test the mineral oil content in the oil vapour was measured and these results are given
in Table 4.11.
oil type An An Au Au Bn Bn
Temp range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Simulation time (min) 30 60 30 60 30 60
µg/m3 µg/m3 µg/m3 µg/m3 µg/m3 µg/m3
Naphthalene 0.78 4.1 0.36 8.3 8.9 93
Acenaphthylene < 0.02 < 0.01 < 0.02 < 0.02 < 0.02 0.14
Acenaphthene 0.02 0.03 0.02 0.18 0.03 0.82
Fluorene 0.04 0.07 0.03 0.26 0.04 0.68
Phenanthrene 0.10 0.08 0.10 0.54 0.15 1.9
Fluoranthene 0.03 0.03 0.02 0.05 0.03 0.17
Pyrene 0.03 0.02 0.03 0.13 0.04 0.21
Benzo[a]anthracene < 0.02 < 0.01 < 0.02 0.01 < 0.02 0.11
Chrysene < 0.02 < 0.01 < 0.02 0.02 < 0.02 0.08
Σ PAH's ((semi)-volatile) 0.99 4.3 0.57 9.5 9.2 97
Σ PAH's particle bound < < < < < <
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Table 4.11. Concentration of mineral oil in the oil vapours during each simulation test.
From the results it was found that during cruise a minimum of 88% and a maximum of 99% of the total mineral oil
vapour measured is emitted at 375 ± 25°C.
Based on the gas chromatographic analysis, the mass distribution was obtained for each simulation test. Figure
4.13 shows from top to down the mineral oil composition of the liquid oil An, its composition of the vapour for
simulation 1 (20 to 350°C) and its composition of the vapour for simulation 2 (new, 375°C).
Based on the results it was found that the mass distribution at room temperature of the original oil An (Figure 4.14,
top) consists of alkane chains in the range of C24-C50. After 30 minutes of heating from 20 up to 350°C the
composition of mineral oil in the vapour remains unchanged and is comparable with the mineral oil chains found in
the original oil at room temperature (Figure 4.14, middle). After heating at a temperature of 375˚C, small changes in
composition of the oil are observed in the chromatogram (Figure 4.14, bottom). There is an increase of relative low
boiling point compounds during the simulation test at 375˚C. Additionally unidentified complex mixtures (UME) are
formed beneath the C24-C50 peaks.
The results of the mineral oil analysis for used oil A (Au) gave at room temperature similar results as were found for
new oil A (An).
oil type An An Au Au Bn Bn
Temp range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
experimental duration (min) 30 60 30 60 30 60
Component mg/m3 mg/m3 mg/m3 mg/m3 mg/m3 mg/m3
Mineral oil 18 420 2,0 380 31 230
Mineral oil expresssed as a percentage (%)
of the total amount of mineral oil measured 4 96 1 99 12 88
during the simulation test
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Figure 4.14. Gas chromatograms of three experimental stages of the new oil A (An) showing the composition of the
mineral oils found per stage. Top: at room temperature. Middle for simulation 1 (20 to 350°C). And at the bottom for
simulation 2 at (375 ± 25)°C.
Based on the results, it was found that the mineral oil composition for oil Bn is comparable with the composition
found for oil An. Also for oil Bn the alkane chains have a range of C24 – C50, with the exception of one peak found
around C21. Additionally the ratio of the alkane chains differs between the two oils. Figure 4.15 shows the
chromatograms of oil An and oil Bn.
2019.51918.51817.51716.51615.51514.51413.51312.51211.51110.5109.598.587.576.565.554.543.532.521.510.50
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W 0
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A 1
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0 C2
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0
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C3
4
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0
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OF
F
RT [min]
µV MO120189.DATA
2019.51918.51817.51716.51615.51514.51413.51312.51211.51110.5109.598.587.576.565.554.543.532.521.510.5
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MO120165.DATAµV
Liquid oil
Type An
Oil vapour
Type An
20-350°C
Increase of
components
formed during the
simulation test at
375°C
Formation of
Unidentified
complex mixture
mixture
Oil vapour
An
375±25°C
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Figure 4.15. Gas chromatograms showing the composition of both new oils. Top: oil An. Bottom: oil Bn.
VOCs in oil vapour 4.4.5
The results of the VOC analysis in the oil vapours are summarized in Table 4.12.. The complete set of results are
presented in Appendix 4 of this report.
2019.51918.51817.51716.51615.51514.51413.51312.51211.51110.5109.598.587.576.565.554.543.532.521.510.5
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6
C28
C30
C32
C34
C36
C38
C40 C4
4
TI
OF
F
RT [min]
µV MO120183.DATA
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OF
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µV MO120184.DATA
Liquid oil
Type An
Liquid oil
Type Bn
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Table 4.12. Summarized results of VOCs found in the simulation tests. Here the sum per group of compounds is
presented. Additionally some various compounds are given, that do not belong to the given groups
<: below detection limit
>: minimum concentration found, chromatographic peaks out of scale and above linearity.
(semi-quantitative analysis)
Based on a selection of the obtained results (aromatics, ketones, and esters) it was found that the emission of both
oils differ at both simulations ‘ground level to top of climb (20 to 350°C) and subsequently cruise speed at (375 ±
25)°C. For example the total VOCs (aromatics, including benzene, toluene and xylene isomers) was for the oil An
and oil Bn at simulation 1 (i.e., 20 to 350°C), 36 µg/m3 and 48 µg/m
3 respectively. These concentrations increase
drastically for these oils at cruise simulation (375 ± 25°C), 1.279 and 3.098 µg/m3 respectively. Similar results were
observed for other volatiles as alkanes and various compounds. These findings of the presence of high
concentrations of compounds at cruise simulation may indicate emissions do take place at higher temperatures.
Besides the quantitative analysis of VOCs, identification of remaining peaks were carried out for those peaks with
sufficient intensity. The peaks were identified based on peak deconvolution with AMDIS followed by a target library
search. The target library contains over a 1000 compounds with spectra and retention indices. Identification was
based on the NET match factor, a combination of the match factor and the retention index, with a minimum NET
match factor of 80. Compounds that were not identified with the target library search were tentatively identified by a
search in the NIST library with a minimum match factor of 80.
Table 4.13 shows a summary of identified compound groups and the amount of different compounds per group
detected in the oil vapours. The complete set of individual identified compounds are presented in Appendix 4.
Oil type An An Au Au Bn Bn
Temperature range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Test duration (min) 30 60 30 60 30 60
Component µg/m3 µg/m3 µg/m3 µg/m3 µg/m3 µg/m3
Σ Aromates 36 1279 6.0 1120 48 3098
Σ Alkanes 318 107 8.8 42 106 88
Various components
2-hexanone 257 > 10000 47 > 8500 298 > 19000
cyclohexanone < > 1000 < 508 < > 2400
phenol 62 136 18 164 239 883
3-methylphenol 289 856 38 1395 503 2467
octanal < 321 10 461 66 1318
MIBK < < < 117 < <
dimethylphthalate 6.0 < 6.9 < 6.7 <
Σ components 968 > 2700 134 > 3806 1266 > 7855
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Table 4.13 Number of individual compounds identified in oil vapour.
Aldehydes in oil vapour 4.4.6
The results of the aldehyde analysis in the oil vapours for the different simulations are presented in Table 4.14.
Table 4.14. The concentration of aldehydes found in the oil vapour during the different simulations.
<: below detection limit
Relative high concentrations were found in the oil vapours for formaldehyde and acetaldehyde. The highest
concentrations of total aldehydes were found for the new oils in the beginning of the simulation test (20 to 350°C).
However, the used oil A (Au) showed another pattern, whereby the highest concentration of aldehydes was
measured at 375±25°C.
It has to be stated, that the sampling of aldehydes was affected by the matrix of oil vapours, resulting in
breakthrough of aldehydes. DNPH cartridges turned from yellow to brown colour (see Figure 4.16), and no DNPH
was left on the desorbed cartridge. Based on this fact, it can be concluded that due to matrix effects, the results of
the aldehydes sampled are probably underestimated concentrations, results must be considered as indicative.
Compound groups Number of individual
compounds identified
in oil vapour
Aldehydes 13
Ketones 23
Alkenes 15
Organic acids 13
Esters 11
Alcohols 2
Furanes 8
Various components 8
Total compounds observed 93
Oil type An An Au Au Bn Bn
Temperature range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Test duration (min) 30 60 30 60 30 60
Component µg/m3 µg/m3 µg/m3 µg/m3 µg/m3 µg/m3
Formaldehyde 2953 465 670 2900 5000 3300
Acetaldehyde 2555 72 1900 920 6100 162
Propionaldehyde 190 10 < 490 1300 420
Crotonaldehyde 51 93 32 450 260 330
n-Butyraldehyde 129 14 470 1300 900 30
Benzaldehyde < < < 42 25 70
iso-Valeraldehyde 11 28 39 100 < <
n-Valeraldehyde 98 99 68 480 300 62
m-Tolualdehyde 140 100 87 220 170 110
p-Tolualdehyde < < 46 44 < <
Hexanal 260 76 30 < 180 300
Σ aldehydes 6387 957 3342 6946 14235 4784
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Figure 4.16. Left: DNPH cartridges after sampling oil vapours at 375°C. Right: DNPH cartridges
after sampling the blank system at 375°C.
Particle number and carbon monoxide in oil vapours 4.4.7
Particle number concentrations (PNCs)
Particle number concentrations (PNCs) were measured during each simulation test. Measurements were
performed with a cut-off of 4 nm to 1000 nm particle diameter and data points were measured each second.
Concentrations of particles were calculated in particle numbers per cm3. For each simulation test mean, minimum
and maximum PNC were calculated over the following four time intervals:
1. Start simulation from 0 - 13 minutes; temperature range 20 to 180°C
2. Simulation from 13 - 31 minutes; temperature range 180 to 350°C
3. Simulation from 0 - 30 minutes; temperature range (375 ± 25)°C
4. Simulation from 30 - 60 minutes; temperature range (375 ± 25)°C
Tables 4.15 - 4.17 shows the results of the PNCs in the oil vapours during the simulation tests.
Table 4.15. PNCs in the oil vapour of new oil A (An) measured in the simulation test and presented for the four
intervals
Oil type An An An An
Temperature range (˚C) 20-350 20-350 375±25 375±25
Test interval (min) 0-13 13-31 0-30 30-60
#/cm3 #/cm
3 #/cm
3 #/cm
3
Mean PNC 86 725200 83278 2156
Minimum PNC 37 237 6656 1088
Maximum PNC 760 1584000 357520 6656
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Table 4.16. PNs in the oil vapour of used oil A (Au) measured in the simulation test and presented for the four
intervals.
Table 4.17. PNCs in the oil vapour of new oil B (Bn) measured in the simulation test and presented for the four
intervals.
A visual presentation on the PNCs is another way to demonstrate what is really happening during the total
simulation and thus on what is happening during the four intervals where the temperature is raising from room
temperature to 350°C and the steady state at (375 ± 25)°C. These results are presented in Figure 4.17.
Oil type Au Au Au Au
Temperature range (˚C) 20-350 20-350 375±25 375±25
Test interval (min) 0-13 13-31 0-30 30-60
#/cm3 #/cm3 #/cm3 #/cm3
Mean PNC 646 47512 87815 290696
Minimum PNC 191 4934 14600 97978
Maximum PNC 4130 72665 305470 416230
Oil type Bn Bn Bn Bn
Temperature range (˚C) 20-350 20-350 375±25 375±25
Test interval (min) 0-13 13-32 0-30 30-60
#/cm3 #/cm3 #/cm3 #/cm3
Mean PNC 1610 421031 75385 13215
Minimum PNC 253 45061 14940 8235
Maximum PNC 45061 773820 286330 23677
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Figure 4.17. The PNCs as measured for the oils during the simulation tests and given for the four defined time
intervals. Two intervals are within the heating from room temperature to 350°C and two intervals are within the
steady state at 375 ± 25°C.
Based on the graphs, it is evident that PNCs are rising as the emissions of oil increases. It is found that during
interval 1, i.e., from the start of the simulation test until 13 minutes after start, the oil warms up to approximately
180°C; here PNCs are relative low. However, after 13 minutes the temperature of the oil is increasing for interval 2,
i.e., within a time frame for 17 to 20 minutes to 350 and 375˚C respectively; this results in the production of relative
high PNCs.
Furthermore for all the simulation tests except for used oil A (Au) a decrease in PNCs was found after reaching 30 -
40 minutes of heating ( 350 to 375˚C) despite of adding dropwise fresh oil to the reaction chamber.
Based on the above, it can be concluded that heating 25 g fresh (unused) oil results in an emission of a total
volatile fraction within a relative short time.
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Additionally, the PNCs found for the used oil in the time interval 2 (from 13 - 32 minutes after start) differ from the
results of the new oils, where high PNCs were found. In the total time interval from 0 - 30 minutes the PNCs of the
used oil were rising due to the start of the dropwise adding of oil into the reaction chamber.
Carbon monoxide, CO
Carbon monoxide was continuously measured during the simulation tests.
Data points were measured each 5 seconds. Concentrations of CO were calculated in mg/m3. For each simulation
test mean, minimum and maximum
CO concentrations were calculated using intervals similar to those of the PNCs:
1. Start simulation from 0 -13 minutes; temperature range 20 to 180°C
2. Simulation from 13 - 31 minutes; temperature range 180 to 350°C
3. Simulation from 0-30 minutes; temperature range (375±25)°C
4. Simulation from 30-60 minutes; temperature range (375±25)˚C
Tables 4.17 to 4.19 presents the results of the CO concentration in the oil vapours during the simulation tests for
the given four intervals.
Table 4.17. The CO concentration observed in the oil vapour of new oil A (An) as measured in the simulation test
and presented for the four intervals.
Oil type An An An An
Temperature range (˚C) 20-350 20-350 375±25 375±25
Test duration (min) 0-13 13-31 0-30 30-60
unit mg/m3 mg/m3 mg/m3 mg/m3
Mean CO concentration 1.1 16 1432 1816
Minimum CO concentration 1.1 1.1 150 680
Maximum CO concentration 1.2 200 1891 2006
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Table 4.18. The CO concentration observed in the oil vapour of used oil A (Au) as measured in the simulation test and presented for the four intervals.
Table 4.19. The CO concentration observed in the oil vapour of new oil B (Bn) as measured in the simulation test
and presented for the four intervals.
Interval 1: at the start of the simulation tests, CO concentrations are approx. 1 mg/m3 and they can be seen as a
usual background concentration levels for indoor air.
Interval 2: after 13 minutes the temperature of the oils are further increasing within a timeframe of 17 - 20 minutes
to an oil temperature of 350˚C and 375˚C respectively, resulting in a fast increase of a mean CO
concentration of 19 mg/m3 with maximum concentrations of 263 mg/m
3.
Interval 3 and 4: During the following 60 minutes, now the temperature remains at 375˚C while oil is dropwise
added to the vessel, the CO concentration is increasing severe. Now high levels up to 1816
mg/m3 were observed.
Based on these results, it may be concluded that from the start of the simulation test until the oil has reached 180
˚C hardly any emission of CO arises. However, it appears that following the increase of temperature of the oil from
180- 375˚C, CO emissions are formed due to incomplete combustion of the oil.
The conclusion given above can be illustrated with the graphs of the CO concentration observed for the given four
intervals (Figure 4.18).
Oil type Au Au Au Au
Temperature range (˚C) 20-350 20-350 375±25 375±25
Test duration (min) 0-13 13-31 0-30 30-60
mg/m3 mg/m3 mg/m3 mg/m3
Mean CO concentration 1.3 17 676 749
Minimum CO concentration 1.2 1 2 1
Maximum CO concentration 1.4 155 909 909
Oil type Bn Bn Bn Bn
Temperature range (˚C) 20-350 20-350 375±25 375±25
Test duration (min) 0-13 13-31 0-30 30-60
mg/m3 mg/m3 mg/m3 mg/m3
Mean CO concentration 1.2 19 630 840
Minimum CO concentration 1.1 1.2 2.2 11
Maximum CO concentration 1.3 263 882 931
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Figure 4.18. The concentration of the carbon monoxide (CO) per interval as measured in the oil vapours measured
during the simulation tests. For all test counts that the CO concentration increases per interval, thus due to raising
temperature. Upper: new oil A (An); middle: used oil A (Au); lower: new oil B (Bn).
4.5 Limitations for upscaling of the results
The work presented in this report is based on a simulation of a worst-case scenario, i.e., under typical laboratory
conditions creating contaminated bleed air from leaked oil into a cabin. Therefore, boundaries have been set, in
order to assure that all experiments could be carried out within the limit of time given for this research.
The conditions were based on the following main boundaries:
- Exposure temperature of the applied oil resulting in the emission of (semi)volatiles (approx. 375°C);
- Time of exposure: only two time profiles have been applied, i.e. (1) ground level to top of climb and (2)
cruise.
1
16
1432 1816
1
10
100
1000
10000
10:50-11:03 11:03-11:21 11:21-11:51 11:51-12:27
Car
bo
n m
on
oxi
de
(m
g/m
3)
Time period of simulation test
An
Mean COconcentrationover timeperiod
1
17
676 749
1
10
100
1000
10:19-10:32 10:32-10:50 10:50-11:20 11:20-11:54
Car
bo
n m
on
oxi
de
(m
g/m
3)
Time period of simulation test
Au Mean COconcentrationover time period
01
19
630 840
01
10
100
1.000
10:35-10:48 10:48-11:07 11:07-11:37 11:37-12:07
Car
bo
n m
on
oxi
de
(m
g/m
3)
Time period of simulation test
Bn Mean COconcentrationover time period
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- Flow of the stream of fresh and contaminated air resulting in a simulated ventilation rate within the
exposure chamber where the contaminants are sampled from.
For the experiments no differentiation in conditions was made related to different type of engines and engine
power, It has been known, that engine conditions may influence any (semi)volatile emission.
Laboratory simulation has been performed without the use of an ECS/PACK.
For the simulation experiments carried out with the emission chamber, only one flow rate is applied causing a
ventilation rate of about 6.5 h-1
.
Based on the fact that the reciprocal of the concentration of a certain contaminant has a linear relation with the
ventilation rate and sampled in a steady state situation, extrapolation of the results is therefore not immediately
possible. For extrapolation, sampling of contaminants during a steady state, and thus not during a floating state,
using at least three measuring points with a different ventilation rate would be needed.
Therefore it is not meant that results of our work can be up-scaled, neither be related to typical aircraft and
engines, and have to be seen as a worst case scenario performed under laboratory conditions only.
4.6 Conclusions
Based on the chemical characterization in the oils itself and in the emissions of oils performed in the simulation
tests for oil A (new and used) and oil B (new), conclusions were established and are presented below.
Performance of the test
The simulation test contains two stages simulating the start of the engine to top of climb (time frame of the
simulation was 30 minutes) and a steady state period for 60 minutes. During steady-state oil was added dropwise.
The simulation tests were kept under controlled and comparable conditions for each oil. At the start of the
simulation test the temperature of the oils were approx. 21˚C and within 30 minutes the oils reached a temperature
of 350°C. During the next 60 minutes of the simulation test the temperature of the oils amounts (375 ± 25°C).
Performance of the oils
The % loss in weight for the used oil A (Au) is significant lower than for the new oils A (An) and oil Bn, while the
settings of time, temperature and oil mass applied for the simulation test were set equally to each other.
It may be concluded that the used oil contains less semi volatile- and volatile oil fractions compared to the new oils
and thus resulting in less evaporation.
TCPs
Based on the analysis in the original oils it was found that in all oils the tri(m,m,m,)-, tri(m,m,p)-, tri(m,p,p)- and
tri(p,p,p)-cresyl phosphate were detected. Tri(o,o,o)-cresyl phosphate was not detected in the oils.
The mass percentage of TCP calculated from the analysis of the oils corresponds with the oil specifications
described in the MSDS sheets from the suppliers.
Based on the analysis of the oil vapours it was found that the four isomers of TCP were detected in all oil vapours
in the same composition as was found for the original oils. Tri(o,o,o) cresyl phosphate was not detected in the oil
vapours.
From the simulation test it was found that the oils heated at a steady state of 375 ± 25°C emits more TCPs than
compared to the simulation whereby the temperature is heated up from 20 to 350°C.
The simulation test shows a good correlation between the composition of TCP isomers found in the original oil and
in the oil vapours at different temperatures. Comparison of new oil A (An) and used oil A (Au) shows no significant
differences in composition of the 4 isomers.
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PAHs
The results of the analysis of PAHs in the vapour shows more numbers of PAHs as were found in the original oils.
This could imply the follow hypotheses:
There is a possible formation of PAHs during heating of the oil and due to incomplete combustion,
PAHs found in the vapour may originate from small concentrations in the original oil (concentrations below the
detection limit).
Most of the PAHs found in the vapour of the used oil A (Au) could not be found in the used (liquid) oil. This implies
that possible formation of certain PAHs occurred during (incomplete) combustion and that they do not remain in the
oil but are released by its vapour.
Oil Bn contained the highest amount of PAHs. The sum of PAHs found in oil Bn was about 10 to 20 times higher
compared to oil A (for both new and used oil).
Mineral oil
Based on the results it was found that the mass distribution at room temperature of the original oil An (Figure 4.14,
top) consists of alkane chains in the range of C24-C50. After 30 minutes of heating from 20 up to 350°C the
composition of mineral oil in the vapour remains unchanged and is comparable with the mineral oil chains found in
the original oil at room temperature (Figure 4.14, middle). After heating at a temperature of 375˚C, small changes in
composition of the oil are observed in the chromatogram (Figure 4.14, bottom). There is an increase of relative low
boiling point compounds during the simulation test at 375˚C. Additionally unidentified complex mixtures (UME) are
formed beneath the C24-C50 peaks.
The results of the mineral oil analysis for used oil A (Au) gave at room temperature similar results as were found for
new oil A (An). Based on the results, it was found that the mineral oil composition for oil Bn is comparable with the
composition found for oil An. Also for oil Bn the alkane chains have a range of C24 – C50, with the exception of one
peak found around C21. Additionally the ratio of the alkane chains differs between the two oils. Figure 4.15 shows
the chromatograms of oil An and oil Bn.
VOCs Based on a selection of the obtained results (aromatics, ketones, and esters) it was found that the emission of both
oils differ at both simulations ‘ground level to top of climb (20 to 350°C) and subsequently cruise speed (375 ±
25°C).
The total VOCs (aromatics, including benzene, toluene and xylene isomers) for oil A and oil B at simulation one (20
to 350°C) amounts 36 µg/m3 and 48 µg/m
3 respectively. These concentrations increased drastically at cruise
simulation at 375 ± 25°C,1.279 and 3.098 µg/m3 respectively. Similar results were observed for other (various)
volatiles.
Aldehydes
High concentrations were found in the oil vapours for formaldehyde and acetaldehyde. The highest concentrations
of total aldehydes were found for the new oils in the beginning of the simulation test (20 to 350°C). Used oil A (Au)
showed the highest concentrations at 375±25°C.
Due to matrix effects, the results of the aldehydes sampled at 375 ± 25°C were unreliable and results must be
considered as indicative.
PNCs
PNCs are rising as the emissions of oil increases. In order to have a closer look on the emission of PNCs, four time
intervals have been defined for the two simulation tests.
1. Start simulation from 0-13 minutes; temperature range 20 to 180°C
2. Simulation from 13-31 minutes; temperature range 180 to 350°C
3. Simulation from 0-30 minutes; temperature range 375 ± 25°C
4. Simulation from 30-60 minutes; temperature range 375 ± 25°C
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From the start of the simulation test until 13 minutes after start, the oil still warms up at approximately 180°C: PNCs
are relative low.
After 13 minutes the temperature of the oils is increasing within a time frame of 17 - 20 minutes to an oil
temperature of respectively 350 and 375°C. Now relative high PNCs were observed.
For all simulation tests except for used oil A (Au) a decrease in PNCs was found after reaching 30 - 40 minutes of
heating (350 to 375°C) despite of adding dropwise fresh oil to the reaction chamber.
It can be concluded that heating 25 g fresh (unused) oil results in an emission of a total volatile fraction within a
relative short time.
Carbon monoxide (CO)
It may be concluded that from the start of the simulation test until the oil has reached 180°C hardly any emission of
CO arises. It appears that following the temperature of the oil from 180 to 375°C, CO emissions are formed due to
incomplete combustion of the oil.
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5 Task 3: Chemical characterization of the oils after pyrolysis
5.1 Background
Pyrolysis of oils during flight will result in a multi-component mixture with variable concentrations. Even the
selectivity of 1-D chromatography combined with mass spectrometry is not always sufficient for full resolution and
elucidation of all compounds. In that case additional selectivity is required. We therefore proposed to apply
comprehensive GC-MS (GCxGC-MS) to such mixtures. This technique allows the user to fully benefit from the
separating potential of the combined power of two independent gas chromatographic columns. In short, this means
that it is now possible to increase selectivity to investigate and quantify even the most difficult samples, i.e., multi
component mixtures like oils and pyrolysis products thereof. It is also possible to investigate low abundant
compounds (i.e. present in low concentrations) in a very intense multi component mixture.
In comprehensive GC-MS, a normal length separation column is used for pre-separation of the analytes, and a
very short and very thin (internal diameter) separation column is used for a second dimension separation. The
addition of a mass spectrometer (e.g., a Time-of-Flight Mass Spectrometer (ToF-MS) multiplies the capabilities in
terms of increased flexibility and the possibility to identify the individual peaks/compounds, yielding a higher
selectivity. Furthermore, automated data analysis allows appropriate pattern recognition. ToF-MS encompasses a
much higher data acquisition speed than other MS configurations, making it an excellent asset for identification of
oil fume compounds. Hypothetically, a large number of presumed and suspected neurotoxic compounds are
present in jet oil fumes. Extracting and analysing these compounds from such a complex matrix is demanding due
to the large number of interfering compounds, some present at relatively much higher concentrations, which could
jeopardize the analysis. Therefore, multi-dimensional techniques such as GCxGC-ToF-MS seem to be most
promising for discriminating small amounts of toxicologically interesting compounds in the presence of high
abundant compounds (Gregg et al., 2006).
A schematic overview of a 2D GC approach is shown in Figure 5.1. After a primary separation, the outlet of the first
column is injected onto a secondary column. The outlet of the secondary column is attached to an appropriate
detector, in this case a ToF MS, allowing on line identification of the compounds eluting from the column, which can
be represented in a 2D contour plot, or a 3D plot.
Figure 5.1. Schematic representation of the 2D and 3D viewing options of modulated chromatograms.
So, briefly, in the current set of pyrolysis experiments oil was pyrolysed in an oven in which samples can be
exposed for a longer time period (hours) to a constant temperature, under various atmospheres under inert
conditions, ambient air or mixtures thereof. Over time, vapours generated were sampled over time using TenaxTM
tubes, packed with appropriate adsorbent resin (e.g., TA) at appropriate time points.
After sampling, the TenaxTM
Tubes were analysed on the GCxGC-ToF-MS system for the presence, and
identification of pyrolysis products in a selected set of oils.
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5.2 Methods and materials
Experimental design 5.2.1
Three engine jet oils were characterized using GCxGC-ToF-MS. In the initial phase, a 2D GCxGC-ToF-MS profile
of the oils was obtained. This was followed by characterization of samples obtained from fumes at preselected time
points during heating the oil in an oven without oxygen (thermolytic profile) and with oxygen (pyrolytic profile) at a
temperature vs time profile, resembling in-flight oil heating. Separated peaks were identified using a chemical
library, and several comparisons were made to obtain similar and unique compounds in the oils.
Basic profiling of turbine oil with GCxGC-ToF-MS 5.2.2
Three turbine jet engine lubrication oils (An, Au and Bn) were diluted in n-Hexane (for pesticide residue analysis,
Sigma-Aldrich) to an approximate concentration of 100 µg/ml. The samples were analysed on an Agilent, Leco,
GCxGC combined with a ToF-MS. First dimension separation was performed on a standard non-polar VF-5ms
column (50m x 0.32mm x 0.4µm). Second dimension separation was performed on a semi polar VF-17ms column
(1m x 0.1mm x 0.1µm). The dual stage modulator was set to a modulation time of 10 seconds, and liquid nitrogen
was used as coolant. The GC oven was initially held at 45°C for 1 minute and then programmed to increase at
6°C/min to 360°C; after which the final temperature was held for 5 minutes. The second dimension oven as well as
the hot jet had a first dimension oven offset of 10 degrees following the same ramped program. Detection was
performed with a Leco Pegasus IV Time of Flight mass spectrometer with an EI source. Scan ranges were m/z 40
to 550 with an acquisition speed of 100 Hz; detector voltages were 1600 volts. The collected spectra were
deconvoluted and automatically processed. A signal to noise (s/n) ratio > 150 was used as limit for peak detection.
The chromatograms were corrected for background compounds. Peaks were identified (criterion: match factor
>800 /999) using the National Institute of Standards and Technology (NIST) and OPCW library.
Simulation of the flight pattern under nitrogen and oxygen conditions 5.2.3
During a flight the turbine oil undergoes a series of temperature changes caused by changes in engine power. A
basic temperature pattern is shown in Figure 5.2. As an aircraft climbs and descends the air pressure surrounding
the aircraft changes. At cruising altitude the pressure is approximately 266 mmHg. This low pressure results in a
lower oxygen concentration than at ambient pressure at sea level. The estimated oxygen concentration at 30,000
feet is 81 g/m3. Both an oxygen-free and varying oxygen level environment were simulated in an oven (Figure 5.3).
Figure 5.2. Schematic overview of a typical engine temperature profile for a flight duration of approximately 90
minutes. x-axis: time in minutes, y-axis: temperature in °C. The different flight states are labelled and TENAX
sampling points are indicated in red.
The oven was programmed to follow the temperature program shown in Figure 5.2. About 4 ml lubrication oil was
put in a ceramic cup and placed in the tube oven. To simulate inert conditions, a nitrogen flow of 2 l/min was used.
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The oven was heated to 120°C before the ceramic cup was placed in the heated zone of the tube oven. After 30
min the oil was heated within 30 min to 350°C, i.e., the so-called cruising altitude temperature. After one hour at
cruising temperature the oven was cooled within 30 min to 120°C by blowing compressed air into the oven.
Figure 5.3. Pyrolysis oven with a temperature range from 50-1,100°C. The over can operate under air, nitrogen or
other gas
To simulate pyrolysis under oxygenated conditions, a similar temperature pattern was used. During climbing and
descending an average oxygen concentration of 178 g/m3 was used. At cruising temperature, the oxygen
concentration was 81 g/m3. The oxygen concentration was changed by diluting compressed laboratory air with
nitrogen retaining a constant flow of 2 l/min through the tube. In the experiments, the oxygen concentration was
changed at 300°C during climbing and descending.
The exhaust of the quartz tube oven was led through a series of filters to an FTIR. The formation of CO, CO2 and
H2O vapour was to follow the flight pattern and see if any large changes like a combustion occurred. Samples were
drawn from the exhaust using a mass flow controller (5 ml/min) and a vacuum pump. The sample was led through
a glass particle filter to collect high-boiling compounds and oil aerosols. The filtered air was then diluted in nitrogen
10 to 1 and thereof 50 ml/min was drawn over a SGE fritted Optic 3 liner filled with TENAXTM
(ta mesh 60/80) to
trap organic compounds. Samples (TenaxTM
and glass particle filters) were taken during:
The taxi phase (120°C, 20 - 25 min)
At the point of reaching the cruise temperature (335 to 350°C, 58 - 63 min)
At the end of the cruise temperature (350°C, 115 - 120 min)
During descent (240 to 200°C, 135 - 140 min)
Experiments were conducted in duplicate (nitrogen only).
Analysis of pyrolysis samples with GCxGC-ToF-MS 5.2.4
Volatile organic compounds were trapped on the TenaxTM
tubes; 300°C was the maximum desorption temperature
due to TenaxTM
degradation. TenaxTM
samples were analysed on the Optic 3, Agilent, Leco TOF-MS using
desorption. The TenaxTM
liner was placed in an air-cooled injector at 40°C and after 30 s stabilization of flows, the
injector was desorbed splitless with a column flow of 2 ml/min. The injector was programmed to increase at 15°C/s
to the final desorption temperature of 300°C. This temperature was held to the end of the GC run. After a total
desorption time of 2 min, the split vent was opened at 250 ml/min and the column flow was 0.8 ml/min for regular
chemical separation.
First dimension separation was performed on a standard non-polar VF-5ms column (50m x 0.32mm x 0.4µm).
Second dimension separation was performed on a semi polar VF-17ms column (1m x 0.1mm x 0.1µm). The dual
stage modulator was set to a modulation time of 4 seconds, and liquid nitrogen was used as coolant. The GC oven
was initially held at 45°C for 5 minutes and then programmed to increase at 6°C/min to 200°C; then the oven was
programed with 10°C/min to 300°C; after which the final temperature was held for 6.33 minutes. The second
dimension oven was initially held at 55°C for 10 minutes and then programmed to increase at 6°C/min to 200°C;
then the oven was programed with 10°C/min to 310°C; after which the final temperature was held for 2 minutes.
The hot jets had a first dimension oven offset of 10 degrees following the same ramped program. Detection was
performed with a Leco Pegasus IV Time of Flight mass spectrometer with an EI source. Scan ranges were m/z 40
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to 550 with an acquisition speed of 100 Hz; detector voltages were 1600 volts. The collected spectra were
deconvoluted and automatically processed. A signal to noise (s/n) ratio > 150 was used as limit for peak detection.
The chromatograms were corrected for background compounds. The chromatograms were corrected for
background compounds. Peaks were identified (criterion: match factor >800 /999) using the National Institute of
Standards and Technology (NIST) and OPCW library.
Glass particle filters were extracted with 1 ml hexane for ~30 minutes on a rotator. The extract was analysed on
Optic 3, Agilent, Leco TOF-MS. 1 µl was injected (split 50:1) at 350°C, which was kept for the entire run, while the
column flow was 1 ml/min (constant flow). First dimension separation was performed on a standard non polar VF-
5ms column (50m x 0.32mm x 0.4µm). Second dimension separation was performed on a semi polar VF-17ms
column (1m x 0.1mm x 0.1µm). The dual stage modulator was set to a modulation time of 10 seconds, and liquid
nitrogen was used as coolant. The GC oven was initially held at 45°C for 1 minute and then programmed at
6°C/min to 360°C; this final temperature was held for 5 minutes. The second dimension oven as well as the hot jet
had an oven offset of 10 degrees following the same ramped program. Detection was performed as described
above.
5.3 Results and discussion
Basic profiling 5.3.1
The basic oil patterns of the two brands (A and B) were obtained. This yielded the 2D total ion chromatograms
(TIC) shown in Figure 5.4. The patterns of new oil (An) and used oil (Au) were also compared but there were no
significant differences found. Only a set of TCP isomers, 4-octyl-N-(4-octylphenyl)-benzenamine and N-phenyl-1-
naphthaleneamine could be identified in the basic oil patterns. TCP isomer peaks were found in all three oils (green
arrows, Figure 5.4). N-phenyl-1-naphthaleneamine was only found in oil Bn, although it did not visually appear in
the TIC chromatogram, which indicates a low concentration.
Only minor differences were found between the oil An and oil Bn. In the red circle compounds appear to be a chain
of chemically related compounds. In oil Bn more peaks are found in this red circle than in oil An. The peaks have an
increasing retention both in the first and second dimension column, resulting in a drift upwards in the
chromatogram. The peaks could not be identified with a similarity >800/999 compared to the NIST. The mass
spectrum of one of the first peaks in the red circle of oil Bn is shown in Figure 5.5. The similarity is 724/999 and
therefore named as “unknown”; a suggested structure is shown. Most fragments found in the peak are also present
in the library spectrum, but the difference in intensities results in a low similarity. Other compounds in the found
chain exhibit the same fragmentation patterns. Because of low similarity with a library spectrum they were also
identified as unknown. Probably these peaks are part of a large molecule, like a polyester with a characteristic
fragmentation pattern. Fragments that are characteristic to these molecules have m/z 57 and 85.
Figure 5.4. 2D Total Ion chromatogram of oil An and oil Au. x-axis: 1st dimension retention time (seconds), y-axis:
2nd dimension time (seconds). The peak intensity is shown by colour gradient
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Figure 5.5. In the upper panel, the mass spectrum of peak 96 found in oil Bn at retention time 2740 sec, 1.79 sec is
shown. The mass spectrum of the library hit with the best fit (similarity 724/999) is shown in the lower panel.
Overview of peak analysis and identification following pyrolysis 5.3.2
The TenaxTM
samples resulting from the pyrolysis experiments were analysed using 2D GC combined with TOF-
MS. Peaks were identified using the NIST library and automated data processing. After desorption of the TENAXTM
samples the chromatograms were visually inspected. In Appendix 5, 2D GC Tof MS plots of samples obtained
during the pyrolysis of the separate oils under both N2 (duplicate) and O2 conditions at the different representative
flight stages are shown.
An example of a sample analysis is shown in Figure 5.6. A 3D plot of a TenaxTM
sample is shown. The sample was
taken from oil A at cruise temperature (350°C). The first dimension retention time is displayed (0-3800 sec) on the
x-axis, the second dimension retention time is displayed on the y-axis (seconds) and the peak height is displayed
on the z-axis and shown by colour gradation. Figure 5.6 shows a large variety of volatile and less volatile
compounds in a broad intensity range. For example, 634 peaks were found in this sample. From these peaks,
approximately 170 peaks could be matched to a NIST with a similarity >800).
It needs to be taken into account that identification was based only on library similarity, which can be prone to
errors. For example, the circled intense peak was identified as 4-[2-(methylamino)ethyl]-phenol, with a similarity of
845/999. However molecular ion 151 and fragment m/z 107 were not present in the spectrum. Moreover, the
retention time of the peak did not match with the expected retention time of the suggested compound, which leads
to the conclusion that this is a false match. This indicates that the identification of compounds has to be considered
indicative.
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Figure 5.6. 3D plot TenaxTM
, oil An at reach of cruise temperature. x-axis: 1st dimension time (sec), y-axis: 2nd
dimension time (sec), z-axis: peak height shown by color gradient.
The total number of identified compounds per oil is shown in Figure 5.7. Higher numbers of compounds are present
under oxygen conditions (top light parts of the bars) than under nitrogen conditions (lower dark part of the bars). In
particular under O2 conditions, more compounds were found at high temperatures (cruise, 350°C) than at lower
temperatures. During the taxi stage (120°C). the highest number of compounds is generated from heating oil Bn, of
which ~90% is unique compared to oil An. For oil Bn, the number of compounds over the flight stages remains
similar, whereas higher numbers of compounds are found in oil A oil at increasing temperatures under nitrogen
conditions. Remarkably, the used oil contains ~50% of unique compounds compared to new oil, indicating a
substantial change of composition over the time of use. This is also reflected by the generation of higher numbers
of compounds under oxygen conditions during heating of used oil A (Au) compared to new oil A (An). From these
observations, a comprehensive list of 127 compounds was constructed that were identified under both nitrogen and
oxygen conditions, in all oils and during different flight stages (Table 5.6). Note that acetophenone and
benzaldehyde might be formed due to TENAXTM
degradation during sampling, caused by reactive compounds in
the smoke.
Figure 5.7. Total number of identified compounds per oil and flight stage. Samples taken during 4 flight stages: Taxi
(120°C), Reach of cruise (335 to 350°C), end of cruise (350°C) and descent (240 to 200°C) under nitrogen and
oxygen conditions. The shades of the bars represent from bottom to top: the number of uniquely identified
compounds under N2 conditions, overlapping compounds under N2 conditions, uniquely identified compounds
under O2 conditions, and overlapping compounds under O2 conditions.
Comparison of oil A and oil B after pyrolysis under nitrogen 5.3.3
Two brands of turbine oil were tested with a temperature program under nitrogen conditions as described under
methods. Samples were taken at similar moments during the experiment and were visually compared. Figure 5.8
shows oil An (left) and oil Bn (right) during taxi state. It is clearly visible that there are some major differences in the
chromatograms (marked by red circles). Peaks that were found in the orange box were both present in oil An as
well as in oil Bn. Identification matches of compounds found in both oils are shown in Table 5.1.
T a x i R . C r u is e En d C r u is e D e s c e n t
0
2 0 0
4 0 0
6 0 0
# C
om
po
un
ds
Bn
A n
A u
O 2
N 2
U n iq u e
c o m p o u n d s
U n iq u e
c o m p o u n d s
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Figure 5.8. 2D TIC of a Tenax
TM sample during taxi temperature. Left panel oil An, right panel oil Bn. X-axis: 1st
dimension time (sec), y-axis: 2nd dimension time (sec), peak height shown by colour gradient.
Table 5.1. Compounds identified (match >800) in both oils, pyrolysis under nitrogen, ranked by similarity match.
Name CAS
1-Tridecanol 112-70-9
2,4-Dimethyl-1-heptene 19549-87-2
2-Ethylhexyl salicylate 118-60-5
2-Propanol, 2-methyl- 75-65-0
5,9-Undecadien-2-one, 6,10-dimethyl- 689-67-8
Acetophenone 98-86-2
Benzaldehyde 100-52-7
Benzene 71-43-2
Benzene, 1,3-bis(1,1-dimethylethyl)- 1014-60-4
Cyclotrisiloxane, hexamethyl- 541-05-9
Decanal 112-31-2
Diethyl Phthalate 84-66-2
Dodecanoic acid, isooctyl ester 84713-06-4
Heptane, 4-methyl- 589-53-7
Hexane, 2,3,4-trimethyl- 921-47-1
Nonanal 124-19-6
Nonane 111-84-2
Octanal 124-13-0
Octane 111-65-9
Phenol, 4-[2-(methylamino)ethyl]- 370-98-9
Glycerin 56-81-5
l-Pantoyl lactone 5405-40-3
Nonane, 2,6-dimethyl- 17302-28-2
Butylated Hydroxytoluene 128-37-0
In oil An five compounds were found during taxi state that were not found in oil Bn during taxi state (120°C). The
compounds are listed in Table 5.2 sorted on similarity. In oil Bn 70 compounds were identified in duplicate that did
not occur in oil An. The 10 compounds with the highest similarities are listed in Table 5.3.
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Table 5.2. Unique compounds that were identified in oil An and were not found in oil Bn during taxi state under
nitrogen.
Name CAS Similarity
Decane 124-18-5 911
Hexane, 2-methyl- 591-76-4 855
Octane, 1-chloro- 111-85-3 826
Glycidol 556-52-5 820
Decane, 1-chloro- 1002-69-3 812
Table 5.3. Unique compounds that were identified in oil Bn and were not found in oil An during taxi state under
nitrogen
Name CAS Similarity
Pentanoic acid, methyl ester 624-24-8 943
Cycloprop[a]indene, 1,1a,6,6a-tetrahydro- 15677-15-3 929
2-Hexanone 591-78-6 922
1H-Indene, 1-methylene- 2471-84-3 921
Decane, 3,7-dimethyl- 17312-54-8 917
2-Heptanone 110-43-0 915
Naphthalene, decahydro-, trans- 493-02-7 914
Dodecane 112-40-3 913
Undecanal 112-44-7 911
Undecane, 2,6-dimethyl- 17301-23-4 911
Comparison of new oil (An) and used oil (Au) after pyrolysis under nitrogen 5.3.4
The used oil had a high similarity with the new oil when their basic profiles were determined. Both oils were tested
under the same experimental conditions and chromatograms from TenaxTM
samples were visually compared.
Figure 5.9 shows the 2D TIC chromatograms of a TenaxTM
sample taken at the moment cruise temperature was
reached.
Some distinct differences between the profiles of the new oil (An) and the used oil (Au) were found. It seems that in
new oil more high boiling compounds were present. This is indicated by two red circles on the right side of the left
chromatogram. These compounds do not appear in the used oil. On the other hand, the used oil shows a large
group of peaks right in the middle of the chromatogram, which were either not present or in a much lower
concentration in comparison to the new oil. It seems that in the duplicate experiments the same patterns were
found in the chromatograms (Figure 5.10). In the sample taken at reach of cruise in the experiments with the new
oil a 243 compounds were identified, only 95 were also identified in the duplicate experiment. In the experiment
with the used oil 294 compounds were identified in the sample taken at reach of cruise, only 86 were identified in
duplicate. Approximately 35% of the identified compounds were found in the duplicate experiments. This gives an
indication of the bias in the experiments.
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Figure 5.9. 2D TIC Tenax
TM at reach of cruise temperature. Left panel new oil (An), right panel used oil (Au). x-axis:
1st dimension time (sec), y-axis: 2nd dimension time (sec), peak height shown by colour gradient.
Figure 5.10. 2D TIC Tenax
TM at reach of cruise temperature. Left panel duplicate new oil (An), right panel duplicate
used oil (Au). x-axis: 1st dimension time (sec), y-axis: 2nd dimension time (sec), peak height shown by colour
gradient.
In the chromatograms above 21 compounds were identified in the new oil that were not present in the
chromatograms of used oil. On the other hand, 30 compounds were found in the chromatograms of used oil that
were not in the new oil. The 10 compounds with the highest similarities are shown in Table 5.4 and Table 5.5. The
reported compounds were found in duplicate experiments, which makes it more plausible that usage of the oil
leads to formation of these compounds during pyrolysis.
Table 5.4. Compounds found in new oil when the cruise temperature (350°C) was reached that were not found in
used oil.
Name CAS Similarity
Cyclopropane, ethyl- 1191-96-4 917
trans-3-Decene 19150-21-1 911
Decane 124-18-5 909
3-Undecene, (Z)- 821-97-6 892
Heptane, 3-ethyl- 15869-80-4 887
Isopropyl Myristate 110-27-0 876
Decane, 3,6-dimethyl- 17312-53-7 874
Ethanone, 1,2-diphenyl- 451-40-1 874
Cyclopropane, 1,1,2,2-tetramethyl- 4127-47-3 873
Homomenthyl salicylate 52253-93-7 867
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Table 5.5. Compounds found in used oil when the cruise temperature (350°C) was reached that were not found in
new oil.
Name CAS Similarity
2-Ethylacrolein 922-63-4 914
2-Hexanone 591-78-6 911
2-Heptanone 110-43-0 910
Cycloprop[a]indene, 1,1a,6,6a-tetrahydro- 15677-15-3 896
Pentadecane 629-62-9 889
3-Tetradecene, (Z)- 41446-67-7 889
2-Butanone 78-93-3 887
Benzonitrile 100-47-0 886
Tetradecane 629-59-4 885
Pentadecane, 2,6,10-trimethyl- 3892-00-0 879
FTIR analysis of exhaust air, formed during pyrolysis of oil under nitrogen 5.3.5
The formed smoke was analysed on-line by FTIR to monitor changes in the exhaust during the experiment. The
response of oil B under nitrogen is shown in Figure 5.11 (left panel). Changes in oven temperature are also
indicated. The graph displays 4 lines: water vapour (green), carbon monoxide (red), ethane (black) and CO2 (blue).
These compounds could be semi-quantified using the standard calibration curves of the software. Since the smoke
is a mixture of compounds and the absorption bands overlap, the concentration is only an indication. In Figure 5.11
(right) the infrared spectrum at the highest concentration of both CO and hydrocarbons is displayed. Characteristic
abundance peaks are circled and named. Figure 5.12 shows two reference spectra of both ethane and CO2.
Ethane was chosen as a marker for all hydrocarbons because of the absorption band at 2600-3200cm-1
. As shown
in Figure 5.12, the peak shapes of the sample and ethane are not completely similar to one another, which is
caused by interference of other hydrocarbons like nonane and decane. These compounds were also found in the
TenaxTM
samples.
When the oil is placed in a pre-heated oven at 120°C, the concentration of hydrocarbons in the exhaust air starts to
increase. After the oven is heated to approximately 320 to 350°C the concentration rises rapidly, and at the same
time CO is detected by the FTIR. After 20 minutes at cruise temperature the concentrations stabilize, followed by a
progressive decline in concentration. A few minutes after starting cooling of the oven, the concentration of both
hydrocarbons and CO starts to decrease rapidly.
Figure 5.11. Left panel: response on the FTIR during pyrolysis of oil B under nitrogen. X-axis: time (hours), y-axis:
response (%). Lines: water vapour (green), CO (red), ethane (black) and CO2 (blue). Right panel: Infrared spectrum
at highest concentration hydrocarbons.
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Figure 5.12. Sample (grey and black) and reference (red) overlay; on the left panel ethane reference, on the right
panel CO2 reference.
Pyrolysis experiments in the presence of oxygen 5.3.6
A series of experiments was performed with varying oxygen concentrations. Samples were taken at the same time
points as in previous experiments.
After the experiment, the ceramic cup had turned completely black (Figure 5.13), which was not the case when
experiments were conducted under nitrogen conditions. The quartz tube in the oven was covered with a tar like
substance. The cup and tube were both cleaned by heating the oven to 900°C with an 2 l/min air flow through it.
This burned off all organic compounds and cleaned the quartz tube.
Figure 5.13. Pictures of oil B before and after the experiment
When the chromatograms of the samples taken during the taxi state (120°C) under oxygen were compared with the
nitrogen equivalent they seem quite similar to one another (Figure 5.14).
Figure 5.14. 2D TIC chromatogram Tenax
TM sample during taxi temperature. Left panel Oil A under N2, right panel
oil A with O2. X-axis: 1st dimension time (sec), y-axis: 2nd dimension time (sec), peak height shown by colour
gradient
Heating of the oils to cruising temperature (350°C) led to formation of a thick white/grey smoke in the tube exhaust.
Chromatograms of samples taken from this are shown in Figure 5.15. Both chromatograms of oil An and oil Bn
show a high saturation of both the number and concentration of compounds. An overload as found in these
samples led to poor separation and increases the chance of false identification. To permit appropriate separation
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and identification, an apparently much higher dilution was required. As this was not anticipated based on the
outcomes of the nitrogen experiments, this was not performed. In the FTIR measurements, combustion of organic
compounds was detected, most likely responsible for the latter findings.
Figure 5.15. 2D TIC chromatogram Tenax
TM sample reaching of cruise temperature with O2. Left panel oil An, right
panel oil Bn. X-axis: 1st dimension time (sec), y-axis: 2nd dimension time (sec), peak height shown by colour
gradient.
The glass particle filters that were placed in line with the TenaxTM
sample were extracted with 1 ml hexane. The
extracts were analysed with comprehensive GC. Two chromatograms of oil An are shown in Figure 5.16. Samples
were taken when the oven reached cruise temperature under nitrogen and oxygen conditions. The pattern shown
in the left panel looks similar to the chromatograms obtained from the basic profiling experiments discussed earlier.
This leads to the conclusion that the bulk of the collected sample were oil aerosols. Compounds that might have
been formed during the pyrolysis were either not trapped on the glass filter or were suppressed by the amount of
raw oil on the filter. Due to the glass filters the TenaxTM
samples obtained during the nitrogen experiments did not
contain the oil pattern as shown in Figure 5.16 and were therefore relevant and safe to analyse.
The right chromatogram shows oil An at cruise temperature obtained from the experiment with oxygen. The
ambient oxygen concentration in the tube was approximately 81 g/m3. Clearly there are some differences in the two
chromatograms. The right chromatogram shows more volatile organic compounds shown in the green circle. On
the other hand there are less highly boiling compounds present. One of the compounds that were not present in the
nitrogen experiment was pentanoic acid. This compound was found with a library similarity 911/999.
TCP isomers could be identified on filter extracts and not on TenaxTM
samples because of the maximum desorption
temperature. The exact chemical structure of the individual isomers of TCP could not be elucidated, however, as
their mass spectra are almost identical.
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Figure 5.16. 2D TIC chromatogram filter extract at reaching of cruise temperature. Left panel oil An under nitrogen,
right panel oil An under oxygen. X-axis: 1st dimension time (sec), y-axis: 2nd dimension time (sec), peak height
shown by colour gradient
Figure 5.17. 2D TIC chromatogram filter extract at reaching of cruise temperature. Left panel oil Bn under nitrogen,
right panel oil Bn under oxygen. X-axis: 1st dimension time (sec), y-axis: 2nd dimension time (sec), peak height
shown by colour gradient.
FTIR analysis of exhaust air, formed during pyrolysis in the presence of oxygen 5.3.7
Before the oil is placed in the oven a concentration of CO2 is detected by the FTIR due to compressed laboratory
air that is led into the tube. This is in contradiction to the experiments under nitrogen where no CO2 was detected
before the oil was placed in the oven. Figure 5.18 shows the FTIR signal of the flight pattern of oil Bn with changing
oxygen concentrations. During taxi state the signal of ethane, as representative for organic compounds, rises
slightly and levels off before the heating starts. As the temperature rises the response increases; when the oven
reached 300°C the concentration oxygen is reduced by mixing compressed air with nitrogen. At the same time the
response highly increased. CO2 and water vapour are formed indicating a combustion of organic compounds. CO
is also formed which is probably caused by the lack of oxygen resulting in an incomplete combustion. When the
oven temperature reached 350°C both ethane and CO had reached their maximum response. Before the oven had
cooled down, the response of CO, CO2 and water vapour started to decrease continuously. It might be possible
that the ceramic cup was already empty at this point. The ethane response did not decrease at all. Probably the
filters between the exhaust of the tube and the FTIR were completely filled with oil.
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Figure 5.18. Response on the FTIR during a pyrolysis of oil Bn with changing oxygen concentrations. X-axis: time
(hours), y-axis: response (%). Lines: water vapour (green), CO (red), ethane (black) and CO2 (blue).
5.4 Conclusion
The aim was to identify compounds in three oils under a variety of conditions.
In the basic oil patterns, without heating, a set of TCP isomers and 4-octyl-N-(4-octylphenyl)-benzenamine were
found in all three oils (green arrows, Figure 5.4). N-phenyl-1-naphthaleneamine was only found in oil Bn, albeit in
low concentrations.
Heating under nitrogen led to an increase in the number of compounds found, and led to the identification of 24
compounds in the vapour, found in all oils (Table 5.1). A number of compounds was identified unique for either oil
An or oil Bn. In addition, used oil (Au) appeared to contain newly identified compounds compared to unused oil (An),
and a number of compounds originally present appeared to have disappeared during use in an engine jet. This
indicates that during the lifetime of an oil, substantial changes in composition occur.
As it cannot be excluded that oxygen is present in the jet engine, the effect of oxygen addition during pyrolysis was
investigated. These experiments showed that the presence of oxygen led to combustion of the oils, resulting in a
major increase of the number and amount of compounds.
To permit a safety assessment of compounds originating from jet engine oils, a list of compounds identified under
both nitrogen and oxygen conditions, in all oils and during different flight stages was constructed, resulting in 127
compounds (Table 5.6). This list can be used to assess the hazard profile of these compounds using the
Classification and Labelling (C&L) database of the European Chemical Agency (ECHA). As a first step, the
harmonised classifications as well as the main self-classifications by manufacturers are added to the list of
chemicals, as presented in Appendix 6. The classification of a substance is an indication for its toxicity.
The experiments were all performed under atmospheric pressure. In a jet engine the pressures can reach almost
10 bars. This could interfere with the formation and evaporation of organic compounds. On the basis of the physical
appearance of used oil (Au) a combustion of turbine lubrication oil is not likely to occur on a large scale. This would
also result in a thick smoke and a pungent odour in the cabin during flight.
Table 5.6. Compounds found after pyrolysis in all oils, irrespective of experimental conditions
Compound # Name CAS
1 Diethyl Phthalate 84-66-2
2 1-Nonene, 4,6,8-trimethyl- 54410-98-9
3 2-Ethylhexyl salicylate 118-60-5
4 Acetophenone 98-86-2
5 Benzaldehyde 100-52-7
6 Benzene, 1,3-bis(1,1-dimethylethyl)- 1014-60-4
7 Heptane, 4-methyl- 589-53-7
8 Nonanal 124-19-6
9 2,4-Dimethyl-1-heptene 19549-87-2
10 Decanal 112-31-2
11 Dodecanoic acid, isooctyl ester 84713-06-4
12 Heptadecane, 2,6,10,14-tetramethyl- 18344-37-1
13 Octanal 124-13-0
14 Dodecane, 4,6-dimethyl- 61141-72-8
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15 Heptane 142-82-5
16 5,9-Undecadien-2-one, 6,10-dimethyl- 689-67-8
17 Benzene 71-43-2
18 Glycidol 556-52-5
19 Nonane, 2,6-dimethyl- 17302-28-2
20 2-Propanol, 2-methyl- 75-65-0
21 Decane, 2,3,5,8-tetramethyl- 192823-15-7
22 Nonane 111-84-2
23 Octane 111-65-9
24 Phenol, 2,4-bis(1,1-dimethylethyl)- 96-76-4
25 2,5-Hexanediol, 2,5-dimethyl- 110-03-2
26 Acetic acid, octadecyl ester 822-23-1
27 Undecane 1120-21-4
28 2-Heptanone, 4-methyl- 6137-06-0
29 Hexane, 2,3,4-trimethyl- 921-47-1
30 1,2-Benzenedicarboxylic acid, di-2-propenyl ester 131-17-9
31 1-Iodo-2-methylundecane 73105-67-6
32 Isopropyl Palmitate 142-91-6
33 n-Decanoic acid 334-48-5
34 Tridecane 629-50-5
35 TCP isomer 563-04-2
36 1-Propanol, 2-methyl- 78-83-1
37 1-Tridecanol 112-70-9
38 5-Hepten-2-one, 6-methyl- 110-93-0
39 Decane 124-18-5
40 Pentane 109-66-0
41 Pentanoic acid, methyl ester 624-24-8
42 Amylene Hydrate 75-85-4
43 Glycerin 56-81-5
44 Heptanal 111-71-7
45 Heptanoic acid, methyl ester 106-73-0
46 Octane, 3,5-dimethyl- 15869-93-9
47 Phenol 108-95-2
48 Propane, 2-ethoxy-2-methyl- 637-92-3
49 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester 84-69-5
50 2-Butanone 78-93-3
51 Butylated Hydroxytoluene 128-37-0
52 Decanoic acid, 2-ethylhexyl ester 73947-30-5
53 Diazene, dimethyl- 503-28-6
54 Dodecane, 2-methyl- 1560-97-0
55 Methacrolein 78-85-3
56 Pentadecane 629-62-9
57 Benzaldehyde, 4-methyl- 104-87-0
58 Phenol, 3-methyl- 108-39-4
59 2H-Pyran-2-one, tetrahydro- 542-28-9
60 Benzene, (1,1,2-trimethylpropyl)- 26356-11-6
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61 Cyclopropyl carbinol 2516-33-8
62 Hexadecen-1-ol, trans-9- 64437-47-4
63 Hexanal 66-25-1
64 Isobutane 75-28-5
65 Octanoic acid, methyl ester 111-11-5
66 (2-Aziridinylethyl)amine 4025-37-0
67 1,4-Dioxane-2,5-dione, 3,6-dimethyl-, (3S-cis)- 4511-42-6
68 1-Octene, 3,7-dimethyl- 4984-01-4
69 2-Butene 107-01-7
70 Cyclobutylamine 2516-34-9
71 n-Hexadecanoic acid 57-10-3
72 Tridecane, 3-methyl- 6418-41-3
73 1-Octene 111-66-0
74 2(5H)-Furanone, 3-methyl- 22122-36-7
75 Pentanal 110-62-3
76 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester 4376-20-9
77 2,5-Furandione, dihydro-3-methyl- 4100-80-5
78 Benzeneacetaldehyde 122-78-1
79 Dodecanoic acid 143-07-7
80 Methylglyoxal 78-98-8
81 Pentadecane, 2,6,10-trimethyl- 3892-00-0
82 2-Hexanone 591-78-6
83 Hydroxyurea 127-07-1
84 (S)-2-Hydroxypropanoic acid 79-33-4
85 1,3,5,7-Cyclooctatetraene 629-20-9
86 2-Propanone, 1-hydroxy- 116-09-6
87 Butyrolactone 96-48-0
88 Cyclopentanone 120-92-3
89 Dodecane 112-40-3
90 Eicosane 112-95-8
91 Heptadecane, 2,6-dimethyl- 54105-67-8
92 l-Pantoyl lactone 5405-40-3
93 Pentanoic acid 109-52-4
94 Phthalic anhydride 85-44-9
95 trans-3-Decene 19150-21-1
96 Undecanal 112-44-7
97 1-Hexene 592-41-6
98 1H-Indene, 1-methylene- 2471-84-3
99 1-Pentene 109-67-1
100 Acetone 67-64-1
101 Decane, 3,7-dimethyl- 17312-54-8
102 Formamide, N-methyl- 123-39-7
103 Hexane 110-54-3
104 Octane, 1-chloro- 111-85-3
105 1,3,5-Cycloheptatriene 544-25-2
106 1-Hexadecanol 36653-82-4
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107 3-Undecene, (Z)- 821-97-6
108 Benzonitrile 100-47-0
109 Cyclooctane, 1,4-dimethyl-, cis- 13151-99-0
110 Dodecane, 2,7,10-trimethyl- 74645-98-0
111 Heptane, 3-ethyl- 15869-80-4
112 1-Pentene, 2,4,4-trimethyl- 107-39-1
113 1-Pentene, 2-methyl- 763-29-1
114 1-Propene, 2-methyl- 115-11-7
115 2(3H)-Furanone, dihydro-5-methyl- 108-29-2
116 2-Heptanone 110-43-0
117 2-Hexenal 505-57-7
118 2-Pentanone 107-87-9
119 2-tert-Butyltoluene 1074-92-6
120 Acetic acid 64-19-7
121 cis-2-Nonene 6434-77-1
122 Decane, 1-chloro- 1002-69-3
123 Heptanoic acid 111-14-8
124 Isopropyl Myristate 110-27-0
125 Tetradecanoic acid 544-63-8
126 1-Pentene, 4-methyl- 691-37-2
127 2-Cyclopenten-1-one 930-30-3
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6 Task 3: Performance of the chemical characterization and toxic effects of the oils after pyrolysis.
6.1 Introduction
Toxicity of the oil vapour was assessed in an in vitro model of the human lung (human bronchio-epithelial (HBE)
cell line in co-culture with human endothelial cells (HUVEC) using an air-liquid-interface system. The combination
of HUVEC and HBE cells creates a realistic imitation of the human lung barrier. The lung model is exposed to the
vapours via the air, which is representative for the human real-life situation.
Toxicity towards the lung model was assessed using an MTT assay. With this assay an early phase of toxicity in
the lung is detectable. As no acute lung toxicity is reported in the real life situation, this endpoint will be used to
determine a non-lung toxic exposure level which will be used for further experiments.
The fluid underneath the lung barrier is be used as exposure medium for the neurological model. Using this
approach, the neurological model of choice (rat primary cortical neurons) is exposed to the fraction of the vapour
that translocated from the air- to the fluid-compartment. Medium from underneath lung cells that have been
exposed to clean air was used as negative control whereas medium that has been exposed to vapour without a
lung barrier present will be used as positive control.
6.2 In vitro approach – Air Liquid Interface
ALI - Introduction 6.2.1
The pyrolysis products of the turbine engine oil can enter the human body through different routes including via
inhalation and skin contact. Given the observation that aviation turbine oil is released into the aircraft environment
as vapour, the consortium considers inhalation the primary or most pertinent route of exposure. The other routes
can also contribute to the total body exposure; however, primary attention should be paid to the inhalation route.
Considering the nature of the reported health complaints the potential effects of exposure stems from disturbance
of the central nervous system function. Hence, the preferred methodology consists of an integrated in vitro
approach integrating exposure of a (human) in vitro lung model with functional measurements in neuronal
networks.
Most cell-based in vitro lung methods are based on exposure of submerged cell cultures. However, to execute this
type of exposure, vapour and associated compounds need to be collected in a liquid medium. This procedure is
likely to change the chemical identity of the mixture. Therefore, exposure to the complete volatile mixture is
preferred. To achieve this, we used the Vitrocell® Air Liquid Interface (ALI; VITROCELL SYSTEMS GMBH,
Waldkirch Germany) in which cells can be exposed to a stream of freshly generated vapour and its associated
compounds ((Phillips et al., 2005)). Using this ALl system, an in vitro (human) lung model has been exposed to
freshly generated aviation oil vapour. The lung model of choice existed of two cell types of human origin, cultured
on inserts (see Figure 6.1) resembling the human lung. In this model, cells on top of the insert (human bronchio-
epithelial cells) are directly exposed to the air flow, while the endothelial cells that grow on the bottom are in contact
with a fluid compartment. Following exposure, the medium in the fluid compartment will contain chemical and
particulate compounds from the exposure that cross the lung barrier and likely also biological signal molecules from
the lung model. This medium has been used to expose the neurotoxicologically cell model. Appropriate controls
have been included to be able to discriminate between effects induced by the vapour exposure and potential
effects induced by signal molecules from the lung that may affect neurological signalling.
Figure 6.1. Schematic view of the setup of the exposure unit. Cells are grown on membranes with the top being
exposed to the aerosol and the bottom being submerged in culture medium.
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Figure 6.2 Schematic view of the ALI setup.
ALI - Methods 6.2.2
Cell Culture
Human bronchial epithelial cells 16HBE140- cells (16HBE) were kindly supplied by Dr Gruenert (University of
California, San Francisco, USA) and cultured in DMEM/F12 medium supplemented with 10% FCS, 1% penstrep,
2mM L-Glutamine and 2.5 µg/ml fungizone. Human umbilical vein endothelial cells (HUVEC) cells were purchased
from Life Technologies (Thermo Fisher Scientific Inc., The Netherlands), and cultured in M200 supplemented with
5% LSGS and 1% penstrep. The cells were incubated at 37 °C in a 95% humidified atmosphere containing 5%
CO2. Once confluent, the cells were trypsonized (0.05% Trypsin-EDTA) and transferred to a new culture flask. The
medium was renewed once a week. To obtain a co-culture, the cells were cultured on non-coated polyester
Transwell® insert membranes with 0.4 µm pores and a surface area of 4.67 cm2 (6 well plate inserts). HUVEC cells
were seeded at a density of 50.000 cells/cm2 on the basolateral side of the Transwell® inserts. After 3 hours, the
Transwell® inserts were returned to their original orientation and the 16 HBE cells were seeded at a density of
200.000 cells/cm2 on the apical side. The bi-culture was incubated at 37°C in a 95% humidified atmosphere
containing 5% CO2 overnight.
Exposure in the Air-Liquid Interface
After 24h incubation, the inserts were exposed for 4h to the oil vapour from oil A (An) or oil B (Bn) in the ALI system.
To maximize the probability of particle deposition, exposure was performed under high voltage conditions applying
an electrical charge to the particles.
Aerosol generation
Oil was dosed using a motor driven (TSE type S40200, TSE Systems, Inc. Chesterfield USA) syringe with a Schlick
compressed air spray nozzle (SCHLICK Mod.970/5 S 9, Düsen-Schlick GmbH, Untersiemau/Coburg Germany).
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The oil was nebulized into a heated mixing chamber with pre-heated compressed air, controlled by a Mass Flow
Controller (MFC) (Type F201, Bronkhorst Nederland B.V., Veenendaal, the Netherlands). The nebulizer, mixing
chamber and compressed air were heated to improve the nebulization by decreasing the oil viscosity and surface
tension. The oil aerosol then passed through a stainless steel tube heated by two tube ovens (Heraeus type ROK/A
6/30, Heraeus Holding GmbH, Hanau Germany) where the oil is vaporized/pyrolized. The air/oil mixture
temperature was measured inside the tube just before the end of the second tube oven by a thermocouple
(Voltcraft K201 thermometer + Type K inconel 600VV thermocouple). At the exit of the ovens, the air/oil mixture
was diluted and cooled with compressed air controlled by a MFC. The dilution flow is concentric and to the outside
of the air/oil mixture to minimize thermal diffusion losses by shielding it from the cold walls of the connection tube to
the ALI. Prior to entering the ALI the mixture passes through a pre selective inlet, designed to remove particles
larger than 2.5µm. The generator deliverers less air than what the ALI and monitoring instruments demand, so at
the cut off impactor inlet an atmospheric make up air inlet with a HEPA filter allows air to balance the generator and
ALI flows.
Downstream of the pre-selective inlet and before the ALI (Figure 6.3), a sample flow was taken to characterize the
air/oil mixture. A flow splitter (TSI model 3708, TSI Incorporated, Shoreview, MN USA) distributes the air/oil mixture
to inline instruments for measurement of time weighted Particles Mass concentration and vapour composition.
Gravimetric measurement was performed with two parallel filters, one Teflon (R2PJ047; Pall corp., Ann Arbor MI,
USA) and one Glass Microfibre GF/F (Whatman, Maidstone, England) plus a downstream vapour cartridge. A
Sartorius MC-5 microbalance (Sartorius, Goettingen, Germany) was used in controlled relative humidity (40 – 45%)
and temperature (21 – 23°C) conditions to do the mass measurements, the filters were weighed before and after
each exposure. Laboratory and field blanks were used for quality assurance. The filter volume flow was measured
with dry gas meters (Gallus 2000 G1.6, Actaris Meterfabriek B.V, Dordrecht the Netherlands).
To measure mass concentration online, a TEOM (Tapered Element Oscillating Microbalance, Series 1400;
Rupprecht & Patashnick, Thermo Fisher Scientific, East Greenbush, NY USA) was connected.
To measure the number concentration and particle size distribution of particles present in the aerosol, a CPC
(Condensation Particle Counter 3022A, TSI Incorporated, Shoreview, MN USA) and a SMPS (Scanning Mobility
Particle Sizer 3080 including CPC 3788 and Advanced Aerosol Neutralizer 3088) were connected. An aerosol
diluter (Main) was used upstream of the CPC and SMPS to bring the air/oil mixture within their measurement
range.
Figure 6.3. Schematic view of the aerosol generation equipment and the placement of the detection and
measurement instruments.
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Cytotoxicity assessment
Cytotoxicity was measured using the widely used MTT assay, which is based on the conversion of MTS tetrazolium
into coloured formazan by the mitochondria of live cells. Thus, a decrease in cell viability caused by the treatment
can be detected as decreased conversion of the colourless substrate into the coloured product. The effect of the
treatment on cell viability was assessed 24h after exposure to be able to detect delayed toxicity as well.
Result ALI 6.2.3
Cytotoxicity in the human lung model
To determine the exposure level where exposure was maximal with minimum cytotoxicity in the lung model, range
finding experiments have been performed.
Based on the outcome of these experiments, an oil dose of 50ul/h was chosen which resulted in an exposure level
of around 1 mg/m3. All medium in the bottom compartment of the wells was pooled per treatment and frozen in a
clean glass vial awaiting further use in the neurotoxicological experiments.
Figure 6.4. Graph displaying the cell viability data in the human lung model following exposure to the two different
oils. Cell viability data is plotted against mass concentration as measured using the TEOM. Each data point
represents the average cell viability from one experiment with six biological replicates.
Exposure characterization
Using the setup described above, a stable vapour generation was achieved. During the exposures various
parameters were monitored including exit temperature, mass concentration, particle number and size as well as
total carbon content of the gaseous phase. There were slight inter-experiment variations in the exit-temperature
ranging from 330 to 340 °C.
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For all exposures an injection speed of 50 µl/h was used resulting in exposure levels between 0.84 and 1.50 mg/m3
based on TEOM data (Figure 6.5). TEOM data correlated well with the gravimetric measurements on Teflon filters
that were used as control for the electronic measurements (R2=0.92). As no filter recording was obtained in one
experiment, the TEOM data is used to plot the data. Particle size varied slightly with the dose but only in a very
small, well-respirable range (24-46 nm; Figure 6.6). In all exposures the number of particles was comparable
(~1*108 particles/cm3; Figure 6.7). No useful TCA measurements were obtained, most likely because of saturation
at these exposure levels.
Figure 6.5. Graph displaying the correlation between the TEOM recordings and the gravimetric measurements on
Teflon filters. A good correlation between the two measurements was observed (R2 = 0.92).
Figure 6.6. Graph displaying the size of particles formed plotted against the measured dose. The data
demonstrates a correlation (R2 = 0.85) between particle size and dose with all particles well within the inhalable
range (24-46 nm).
0 1 2 3 4
0
1
2
3
4
F ilte r v s T E O M
T E O M (m g /m 3 )
Fil
ter (
mg
/m3
)
0 1 2 3 4
0
2 0
4 0
6 0
S iz e v s D o s e
T E O M (m g /m 3 )
Siz
e (
nm
)
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Figure 6.7. Graph displaying the correlation between the parameters dose and particle number. No correlation (R2
= 0.40) was observed as the particle number was in all experiments ~ 1*108 particles/cm3.
In vitro approach - microelectrode arrays (MEA) 6.2.4
Introduction 6.2.5
Current data indicate that the nervous system is most sensitive to potential effects of pyrolysis products of turbine
engine oil (Winder et al., 2002b, Ross, 2008, Furlong, 2011, Liyasova et al., 2011, Abou-Donia et al., 2013).
Disturbance of neuronal function can induce detrimental short-term as well as long-term consequences, including
cognitive psychological defects as well as impaired neurodevelopment and neurodegeneration.
Earlier studies have shown effects of tricresylphosphates (TCP) on several endpoints involved in neuronal function.
For example, ToCP was recently shown to inhibit voltage-gated calcium channels (VGCC), glutamatergic calcium
signalling and neurite microstructure in mouse embryonic neurons following 1-6 days of exposure (Hausherr et al.,
2014, Hausherr et al., 2016). Yet, effects of pyrolysis products are largely unknown.
Effects of pyrolysis on neuronal function can be assessed in vitro using primary cortical neurons grown on
microelectrode arrays (MEAs). MEAs consist of a cell culture surface with an integrated array of microelectrodes
that allows for the simultaneous and non-invasive recordings of local field potentials at millisecond time scale as
measure for neuronal (network) function (for review, see Johnstone et al.(2010), Figure 6.8).
0 1 2 3 4
1 0 7
1 0 8
1 0 9
P a r t ic le # v s m a s s
T E O M (m g /m 3 )
CP
C (
#/c
m3
)
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Figure 6.8. Maestro 768-channel amplifier with integrated heating system and temperature controller (top) for
recording of neuronal activity. Recordings are performed using 48-well MEA plates (middle left), with each well
containing 16 nano-textured gold microelectrodes (middle, right).Detail of primary rat cortical neurons cultured on
top of the electrode grid (bottom, right) and the resulting signals representing spontaneous neuronal activity
(bottom,left).
Neuronal networks grown on MEAs possess many characteristics of neurons in vivo, including (the development
of) spontaneous activity with bursting (Robinette et al., 2011) and responsiveness to neurotransmitters and
pharmacological agents (Gross et al., 1997, Johnstone et al., 2010, de Groot et al., 2013). Moreover, MEA
recordings have shown consistent reproducibility and reliability across different laboratories(Novellino et al., 2011)
as well as high sensitivity and specificity (McConnell et al., 2012, Nicolas et al., 2014, Valdivia et al., 2014).
Recently, it was confirmed that primary cortical neurons grown on MEAs are responsive to a range of
neurotransmitters and pharmacological modulation of neurotransmitter receptors (Hondebrink et al., 2016) as well
as to toxin-induced modulation of ion channels (Nicolas et al., 2014), highlighting the usability of this system as an
integrated screening tool that is sensitive to a wide range of compounds.
Therefore, effects of pyrolysis products were investigated in vitro using MEA recordings of primary cortical neurons
to assess neuronal network function as an integrated read out, rather than testing all individual processes (modes
of action) involved in neuronal function.
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Methods 6.2.6
Cell culture.
Experiments were approved by the Ethical Committee for Animal Experiments of Utrecht University and were in
accordance with Dutch law. Primary cultures of rat cortical neurons were prepared from postnatal day 0 to 1 Wistar
rat pups as described previously (Nicolas et al., 2014, de Groot et al., 2016, Hondebrink et al., 2016), with minor
modifications. Briefly, pups were decapitated and cortices were rapidly dissected on ice and minced into small
pieces, which were mechanically dissociated by gentle trituration and filtered through a cell strainer (BD Falcon,
100 μm nylon). Cells were resuspended in dissection medium, i.e. Neurobasal-A supplemented with sucrose (14
g/500 mL), 200 mM L-glutamine (Life Technologies, Bleiswijk, The Netherlands), 2.5 mM glutamic acid, 10% fetal
bovine serum (FBS, Life Technologies), and 1% of a solution containing 10 000 units/mL of penicillin and 10 000
μg/mL of streptomycin (Life Technologies). The cell-containing medium was centrifuged for 5 min at 800 rpm and
supernatant was removed. Primary cortical cells were diluted in dissection medium and seeded on 0.1%
polyethyleneimine ([PEI; diluted in borate buffer [24 mM Sodium Borate/50 mM Boric Acid in Milli-Q adjusted to pH
8.4])-coated 48-well MEA plates (Axion Biosystems Inc., Atlanta, USA) at a density of approximately 1 × 105
cells/well for measurements of neuronal activity.
Cells were cultured in a humidified incubator at 37°C and 5% CO2. At day in vitro (DIV) 1, the dissection medium
was replaced by glutamate medium, i.e. Neurobasal-A supplemented with sucrose (14 g/500 mL), 200 mM L-
glutamine, 2.5 mM glutamic acid, 2% B-27 (Life Technologies), and 1% penicillin/streptomycin. On DIV4, glutamate
medium was replaced by FBS culture medium, i.e. Neurobasal-A supplemented with sucrose (14 g/500 mL), 200
mM L-glutamine, 10% FBS, and 1% penicillin/streptomycin (Life Technologies).
Measurements of neuronal electrical activity using microelectrode arrays (MEAs).
Primary rat cortical neurons grow neuronal networks consisting of βIII-tubulin positive neurons and GFAP positive
astrocytes (see Figure 6.9). These primary rat cortical neurons were cultured on 48-well MEA plates, with each well
containing 16 nano-textured gold microelectrodes (40–50 μm diameter; 350 μm centre-to-centre spacing) with four
integrated ground electrodes (see also Figure 6.8). Spontaneous electrical activity was recorded at DIV9-11 at a
constant temperature of 37°C using a Maestro 768-channel amplifier with integrated heating system and
temperature controller (Axion Biosystems Inc.) as described previously (de Groot et al., 2014, Nicolas et al., 2014,
de Groot et al., 2016, Hondebrink et al., 2016). Axion’s Integrated Studio (AxIS 1.7.8) was used to manage data
acquisition. Channels were sampled simultaneously with a gain of 1200× and a sampling frequency of 12.5
kHz/channel using a band-pass filter (200-5000 Hz), resulting in raw data files.
Figure 6.9. Light microscopic image of primary rat cortical cultures (left) and confocal immunofluorescent image
(right) demonstrating the presence and distribution of βIII-tubulin positive neurons (green) and GFAP positive
astrocytes (red) at14 days in culture (DIV14). Blue staining (DAPI) represents nuclei.
MEA plates were allowed to equilibrate in the Maestro for 5-10 min prior to recordings of electrical activity. At
DIV9/10, a 30 min baseline recording of spontaneous activity was made. After this recording the cells were
exposed by adding 55 μL of the test chemical (final dilution 10 and 30x) or 165 μL of the test chemical (final dilution
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4x; minimum dilution possible) and a subsequent 30 min recording was performed directly following the onset of
exposure to determine the acute effect of the test compounds compared to baseline spontaneous activity (paired
comparison). At DIV10/11 neuronal activity was measured again to determine the effects of test compounds
following 24h exposure. Next, effects of test compounds were normalized to time-matched medium controls to
prevent confounding by changes in neuronal activity by ongoing development of cortical cultures over time.
Data analysis and statistics.
For MEA data, raw data files were re-recorded to generate Alpha Map files for further data analysis in
NeuroExplorer® software (Nex Technologies, Madison, USA). During re-recording, spikes were detected using the
AxIS Spike Detector with dynamic threshold detection (Adaptive threshold crossing, Ada BandFlt v2) set at seven
times standard deviation of the internal noise level (rms) on each electrode. The spike count files (Alpha Map files)
were loaded into NeuroExplorer for further analysis of the percentage of active wells (defined as ≥1 active
electrode), the percentage of active electrodes (defined as ≥0.1 spikes/s) per well, and the average mean spike
rate (MSR; spikes/s) per active electrode. Effects of test compounds were calculated as follows: MSRs were
averaged per well (20-31 wells (n) from at least 3-4 independent isolations) and effects of test compounds were
calculated as percentage change compared to baseline. Next, the effects test compounds were expressed (mean ±
SEM from n wells) normalized to time- and dilution-matched medium control wells.
Wells that showed effects two times standard deviation (SD) above or below average are considered outliers (~5%)
and were excluded from further analysis. Effects of test compounds on spontaneous neuronal activity were tested
for significance using unpaired two-sample t-tests. Effects were considered statistically significant if p-values <0.05.
Results 6.2.7
Medium derived from lung epithelial cells exposed via the air-liquid interface (ALI) model was used to expose the in
vitro brain model. Test conditions included medium containing oil An-derived pyrolysis products (4, 10 or 30x
diluted), oil Bn-derived pyrolysis products (4, 10 or 30x diluted), medium derived from lung epithelial cells exposed
via ALI to clean air (4, 10 or 30x diluted), or medium derived directly from the ALI system following exposure to
pyrolysis product but in the absence of lung epithelial cells (‘no insert’; 4 or 10x diluted). Effects of these media on
neuronal activity were compared to time- and dilution-matched medium controls and activity was assessed during
the initial phase of the exposure (acute 30 min exposure) and following 24h exposure (subchronic 24h exposure).
Acute (30 min) exposure.
Neuronal activity in cortical cultures acutely exposed to oil An-derived pyrolysis products showed a modest and
concentration-dependent increase. However, even at the 4x dilution the increase in neuronal activity compared to
medium controls did not reach statistical significance (Figure 6.10, left). Similarly, acute exposure to oil Bn-derived
pyrolysis products induced a modest and concentration-dependent increase in neuronal activity that did not reach
statistical significance (Figure 6.10, right).
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Figure 6.10. Changes in neuronal activity (expressed as percentage of medium controls (n=24-31) induced by
acute (30 min) exposure to different dilutions of oil-derived pyrolysis products following transfer to the air-liquid
interface. None of the effects reached statistical significance. Data are presented as mean ± SEM from 20-26 wells.
Subchronic (24 h) exposure.
Neuronal activity in cortical cultures exposed to oil An-derived pyrolysis products for 24h did not reveal any changes
compared to medium controls (Figure 6.11, left). Similarly, exposure to oil Bn-derived pyrolysis products did not
induce any changes in neuronal activity compared to medium controls (Figure 6.11, right).
Figure 6.11. Changes in neuronal activity (expressed as percentage of medium controls (n=24-31) induced by
subchronic (24 h) exposure to different dilutions of oil-derived pyrolysis products following transfer to the air-liquid
interface. None of the effects reached statistical significance. Data are presented as mean ± SEM from 23-29 wells.
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Direct exposure to pyrolysis products.
Notably, cortical cultures acutely exposed to medium containing pyrolysis products of oil An derived directly from
the ALI system in the absence of long epithelial cells (‘no insert’) showed a significant increase in neuronal activity
compared to medium control (194 ± 23, n=24, p<0.05), but only at the highest concentration (4x diluted; Figure
6.12, left). Acute exposure to medium containing pyrolysis products of oil Bn derived directly from the ALI system in
the absence of long epithelial cells (‘no insert’), did also induce a significant increase in neuronal activity compared
to medium control (163 ± 15, n=23, p<0.05), but only at 10x dilution (Figure 6.12, right). The increase in neuronal
activity in cortical cultures exposed to oil-derived pyrolysis products was no longer visible following subchronic
(24h) exposure (not shown).
Figure 6.12. Changes in neuronal activity (expressed as percentage of medium controls (n=24-31) induced by
acute (30 min) exposure to different dilutions of oil-derived pyrolysis products following transfer to the cell-free (no
insert) air-liquid interface. Data are presented as mean ± SEM from 23-29 wells; *, p <0.05.
Conclusions and discussion 6.2.8
The current data indicate that acute exposure of primary rat cortical cultures to medium containing pyrolysis
products derived from oil An or oil Bn following transfer to an air-liquid interface equipped with lung epithelial cells
does not induce significant changes in neuronal activity (Figures 6.10 and 6.11). The lack of effect at neuronal
activity is also a clear indication for the absence of cell death/cytotoxicity of neuronal cells. Nevertheless, the
highest concentrations tested (4x diluted) do show a trend towards an increase in neuronal activity and it cannot
currently be ruled out that higher concentrations may affect neuronal activity. However, it is practically not possible
to increase the concentration of pyrolysis products in the medium (minimal dilution is 4x, and further increasing the
concentration in the air-liquid interface will induce acute cytotoxicity of the lung epithelial cells).
The observation that neuronal activity is increased when cortical cultures are exposed to medium containing
pyrolysis products following transfer to a cell-free air-liquid interface equipped (no insert, i.e., no lung epithelial cells
present; Figure 6.12) is however suggestive for the presence of neuroactive compounds in the medium.
Apparently, the lung model properly acts as a barrier and prevents that pyrolysis products permeate to a degree
that is sufficient to cause effects.
Due to a lack of insufficient in-flight monitoring data for pyrolysis products it is not easy to compare the in vitro
exposure levels applied in the AVOIL study to real-life exposure as may be expected for cabin air environment
under normal flight conditions.
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Nevertheless, based on measurements performed by TNO (2013b), it is possible to make an estimation as to how
the levels in vitro relate with respect to real-life exposure levels. TNO investigated the concentrations of TCPs in
the oil and in the cockpit air measured under normal flight conditions. The TCP contents and isomeric composition
of the TCPs in the oils used in the aircraft investigated was comparable to the oils tested in the AVOIL experiments.
Therefore, the sum of TCPs (∑TCP) can be used as tracer compound to compare the results from the in vitro
measurements with levels found during the in-flight measurements.
Concentrations reported from the TNO investigation indicate average ∑TCP levels of up to 155 ng/m3 in-flight.
Chemical analysis of the vapour generated for the in vitro exposures displayed ∑TCP levels of 17 (± 3) and 33 (± 7)
µg/m3 for oil An and for oil Bn respectively (Appendix 7) indicating a factor ~100 higher exposure in the in vitro lung
model compared to normal in-flight levels. As an extra dilution factor of 4 is applied in the neurotoxicity experiments
also the exposure level should be divided by 4 when considering the neurotoxicity data. This reduces the difference
between the in vitro exposure levels and everyday exposure levels to a factor ~30-50. Nevertheless, everyday
exposure levels reported in literature range from 0.3 ng/m3 to 50 µg/m
3 (Crump et al., 2011, Denola et al., 2011,
Rosenberger et al., 2013) depending on the aircraft investigated, indicating that great care should be taken when
interpreting these data.
The current data indicate that neuroactive pyrolysis products are present, but that their concentration in the
presence of an intact lung barrier is too low to be of major concern for neuronal function. Moreover, the non-
significant effects appeared to be transient as neuronal activity following 24h exposure is very comparable to
medium controls. This is in line with recent MEA data showing that neuronal function of primary cultured rat cortical
cultures following acute (30 min) exposure to different TCP isomers or commercial mixtures of TCPs is not or only
limitedly affected (Duarte et al., 2016). Similarly, the effects of TCPs appeared to be transient and virtually absent
following 24h exposure. However, when exposure continued up to 48h neuronal activity was markedly decreased
by the majority of TCP isomers and mixtures. It is therefore possible that effects of pyrolysis products develop only
following more prolonged (i.e. 48h and longer). In line with this notion, the effects of TCPs on (glutamatergic)
calcium signalling and neurite microstructure (Hausherr et al., 2014, Hausherr et al., 2016) were also observed
following more prolonged exposure (1-6 days).
So, while the present data indicates limited concern following exposure up to 24h, prolonged exposure to pyrolysis
products may aggravate their potential neurotoxicity. Given the general human (occupational) exposure scenario,
additional research may thus need to focus on prolonged and/or repeated exposure to pyrolysis products. Notably,
such studies may need to focus also on the use of human induced pluripotent stem cell-derived neurons, which are
gradually becoming more accessible for neurotoxicological research (Tukker et al., 2016).
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7 Task 4: Analysis of the human sensitivity variability factor
7.1 Introduction
There are concerns among international governments, pilots, cabin crew and passenger (and other stakeholders of
commercial jet aircraft) about health effects related to the presence of fumes in the air supplied to aircraft cabins.
Pilots, cabin crew as well as passenger have reported a whole range of symptoms (see Appendix 2 and Task 1).
Cabin air quality under routine conditions has only been partially characterized or documented within a limited
number of flights hours, types of aircraft and number of chemical substances (see Appendix 3 and Task 1). There
are, furthermore, no published studies, which describe and quantify air quality in aircraft under abnormal operating
conditions; such as fume events.
The available information about the potential exposure to hazardous substances in the cabin suggests that
environmental factors, including air contaminants, can be responsible for some of the many physical symptoms in
cabin crew and passengers (such as sleeping problems, dizziness, concentration problems, headaches, respiratory
complaints). However, the reported complaints tend to include a much broader range of (un)specific types of
symptoms and within an equally exposed group of individuals, only some people have been found to develop
symptoms, which makes it difficult to cluster the symptoms in a specific disease or syndrome.
It is therefore extremely difficult, if not impossible, to establish a causal relationship between cabin air quality and
the self-reported symptoms which come forward from the different reviews. Extensive reviews (Nagda and Koontz,
2003, Griffiths and Powell, 2012) were unable to attribute any clinical outcomes to specific exposures. Moreover, in
an aircraft there is potential exposure to a large number of substances. Inadequate hazard profiles, mixture
toxicology and the lack of exposure levels allows only for a partial risk assessment. Key issues related to the
difficulty to link the cabin air quality with reported symptoms are the broad variety in reported symptoms, the lack of
systematic routine collected data about health complaints and the lack of clustered symptoms in crew as well as
passengers. For the pilots and other crew members a healthy worker effect might also play a role, and on the other
hand it is also possible that symptoms are underreported in the crew due to the fear of the consequence of a
medical examination, potentially resulting in a “not fit to fly” status.
The variety in somatic complaints and their unspecific nature indicate, at least partly, that we might be dealing with
somatically unexplained physical symptoms; a term which refers to diffuse symptoms after exposure to low doses
of everyday environmental factors.
The main aim of this section is to explore the factors that can contribute to the development of health complaints
caused by exposure to potentially contaminated cabin air and provide potential explanations why a broad range of
(un)specific types of symptoms are reported in only a limited number of individuals exposed. Hereto the available
knowledge on the following topics is summarized:
Genetic differences in metabolism and detoxification (7.2)
The influence of stress/coping strategies on underlying biological pathways leading to health complaints
(7.3)
7.2 Genetic differences in metabolism and detoxification
It is widely accepted that differences exist between humans with respect to chemical sensitivity. Part of these
differences can be explained by ‘normal’ biological variation. Several obvious factors can increase the degree
interindividual differences, including sex, age, general health status and life style factors such as diet, smoking and
drug or alcohol consumption. In addition, specific genetic differences (polymorphisms) can contribute significantly
to the chemical sensitivity of an individual. Such genetic differences can act at different levels to alter an
individual’s chemical sensitivity, for example by altering the bioavailability and/or potency of the chemical of
interest.
The intensity of a toxic effect relates to the concentration and persistence of the ultimate toxicant at its target site.
The concentration of the ultimate toxicant at its target site depends on the relative effectiveness of multiple
processes that affect its delivery (Figure 7.1). In toxicology this is generally referred to as delivery or ADME:
absorption, distribution, metabolism and excretion.
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Figure 7.1. Schematic presentation of the processes that affect the delivery of a toxicant (Klaassen et al., 2013).
ADME largely determines if a particular chemical enters the body, how long it stays in the body and if it can reach
its molecular target. Absorption following oral exposure for example is usually higher in children than in adults.
However, for inhalation exposure which is the most obvious exposure route for CAC, differences in absorption
seem less obvious. With respect to distribution in the body; lipophilic compounds tend to accumulate in fatty
tissues, whereas hydrophilic compounds are usually easily excreted. A person’s fat percentage can thus to some
extent affect the distribution/excretion of a chemical, and consequently alter in theory a person’s chemical
sensitivity. However, the most notable contribution to differences in sensitivity is to be expected from differences in
metabolism.
Metabolism, also known as biotransformation, usually involves transformation to a compound with increased
polarity and potentially altered potency by cytochrome P450 enzymes in phase 1 and subsequent conjugation of
the phase 1 product in phase 2, for example by glucuronidation or sulfation. Interindividual differences in the
activity of phase 1 and/or phase 2 enzymes can thus affect which metabolites are formed and to which extent.
Current knowledge about the enzyme variability with these metabolic pathways in the human population is largely
based on well-studied pharmaceuticals, such as acetaminophen (paracetamol). For example, interindividual
variability in glucuronidation (phase 2 metabolisms) has been indicated to contribute to differences in susceptibility
to acetaminophen intoxication and associated liver injury in humans. Subsequent research demonstrated a
substantial degree of interindividual variability (more than 15-fold) for the responsible phase 2 enzyme UDP-
glucuronosyltransferase (UGT)(Court et al., 2001).
Current knowledge about the enzyme variability with regard to CACs is mainly restricted to ToCP. For ToCP it is
known that cytochrome P450 enzymes are responsible for the formation of a toxic metabolite (bioactivation; also
see (Reinen et al., 2015) ), whereas paraoxonase 1 (PON1) is likely responsible for its detoxification. Individuals
with an enzyme profile that favours bioactivation (cytochrome P450 enzymes) and/or hampers detoxification
(PON1) of ToCP are thus likely to be more sensitive to its toxic effect. Earlier human studies with different
cytochrome P450 enzymes, including 2C19, 3A4, 2D6 and 1A2, indicated a difference in individual constitutive
hepatic activity of approximately 50-100-fold (Rendic and Di Carlo, 1997, Tamminga et al., 1999, Hagg et al.,
2001). An additional 40-fold difference in the constitutive activity of the detoxification enzyme PON1 has previously
been found in humans (Costa et al., 2005). As a result of these interindividual differences in P450 and PON1
enzyme activities, a 4000-fold difference can be expected between individuals expressing a very low and very high
sensitivity (de Ree et al., 2014). Notably, this interindividual difference may be exaggerated due to the experimental
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design, which relies on high substrate concentrations to determine enzyme activity, as a recent study indicated that
the difference in PON1 activity is well within a factor 10 for more realistic substrate concentrations (Coombes et al.,
2014). Yet, this may render a specific subpopulation that could be approximately 1000-fold more sensitive to ToCP.
However, the complete metabolic pathway and the contribution of interindividual variability in the metabolic
enzymes is still largely unknown for the majority of industrial chemicals, including CACs. Nevertheless, similar
differences in sensitivity can be expected for other compounds that rely on cytochrome P450 enzymes for their
metabolism.
In addition to (genetic) metabolic differences, other factors may be involved in the occurrence of interindividual
differences in sensitivity, such as interaction of the chemical of interest with other organ systems and/or targets.
While differences in immune-sensitivity have been suggested, for example in relation to ‘sick-building syndrome’,
there is currently no evidence for interindividual differences in immune-sensitization for ToCP or the different
chemical compounds in CACs. Similarly, genetic differences can alter an individual’s chemical sensitivity directly at
the target such as a specific receptor , enzyme or ion channel. If for example a particular isoform of a receptor has
an altered affinity for the chemical of interest, this may result in altered sensitivity of the individual. Yet, there is
currently no evidence for such interindividual differences directly at the target(s) for ToCP or the different chemical
compounds in CACs.
While the involvement of genetic differences may (partly) explain why some individuals are more sensitive than
others, the question remains whether current exposure levels are sufficiently high to evoke such complaints even in
sensitive individuals.
It should be noted that the range of reported health complaints is very broad and therefore difficult to pinpoint to a
particular CAC. Common acute symptoms include sensory (irritative) effects in eyes and airways as well as
neurological symptoms such as headache. However, although ToCP at sufficiently high concentrations can cause
(acute) respiratory failure by inhibition of acetylcholinesterase, TCPs are generally not considered airway irritants
and it appears that sensory symptoms can be exacerbated by or even due to environmental and occupational
conditions present in the aircraft, such as low relative humidity and low cabin pressure (reviewed in (Wolkoff et al.,
2016)). Moreover, typical symptoms associated with acute inhibition of acetylcholinesterase (SLUDGE: salivation,
lacrimation, urination, diaphoresis, gastrointestinal upset, emesis) are hardly reported.
Next to acute symptoms, there is a wide range of reported chronic health complaints, including fatigue, dizziness,
muscle weakness, nerve pain, tremors and cognitive impairment and even neurodegeneration (reviewed in
(Wolkoff et al., 2016)). While chronic exposure to ToCP can cause organophosphate-induced delayed neuropathy
mediated by inhibition of neuropathy target esterase, these symptoms at best partially match with those reported
following cabin air exposure. It can therefore be questioned if the reported health complaints are related to ToCP
exposure.
Flying is associated with unique exposure conditions including exposure other CACs, radiation, hypoxia and
changes in temperature, gravitational forces and pressure. Whether or not these occupational conditions are
responsible for the reported complaints remains unknown until the complete set of potential chemical exposures is
known, including their exposure levels, resulting internal dose levels, full spectrum of molecular targets (i.e., all
different modes of action) and the related no-effect concentrations.
7.3 The influence of stress/coping strategies on underlying biological pathways leading to health
complaints
A strong body of experimental studies has demonstrated that somatically unexplained physical symptoms occur
when people perceive they are being exposed to environmental stressors irrespective of actual exposure.
Perceived exposure can thus trigger or contribute to the occurrence of symptoms. More specific triggers such as
smell, temperature, stuffy air are hereby relevant and well documented (Bailer et al., 2005, Das-Munshi et al.,
2006). A whole range of explanatory models has been described in the literature for the development and
maintenance of somatically unexplained physical symptoms. Currently a cognitive psychological approach is the
most common (see e.g. Brown (2004)). In this approach symptom-focused attention is considered as central in
both symptoms development and maintenance. Misinterpretation of symptoms, illness beliefs, worry and
rumination, negative affect and personality features are assumed to play an important role in this process.
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One characteristic element often associated with the occurrence of somatically unexplained physical symptoms is
the lack of personal control or learned helplessness, well-known factors to enhance stress reactions, which in their
own right can lead to acute health complaints and long-term health effects (Seligman, 1975, Lazarus and Folkman,
1984). Also in the case of symptoms attributed to environmental factors the lack of control over the exposure is
characteristic (Campbell, 1983). There is a shared notion within the field of environmental stressor that we are
dealing with a generic mechanism. A large body of stress research in the past 40 years shows that environmental
factors are assessed by the individual in terms of threat and control referred to in the stress approach by Lazarus
and Folkman as primary and secondary appraisal. The actual health effects in this process are highly dependent
on how people cope and whether coping options are available. These coping strategies can roughly be subdivided
in active coping and avoidance (Lazarus, 1966, Folkman et al., 1986, Roth and Cohen, 1986, Baliatsas, 2015).
Characteristic for the context of an enclosed environment, such as in an airplane, is that the range of coping
options is very limited and that the work situation is characterized by a range of stressors (McNeely et al., 2014).
Also it is important to emphasize that this mechanism applies to situations where there is actual exposure as well
as perceived exposure. An immediate pathway between exposure and health can co-exists with an indirect
pathway (via beliefs/expectations, personality traits, context and for example media coverage and early
experiences). For several environmental exposures these pathways have been studied in detail such as
environmental noise (van Kamp, 1990), electromagnetic fields (Baliatsas, 2015) and odour (Cavalini et al., 1991,
Winneke et al., 1996).
Two basic approaches can roughly be discerned in the field of stress research: 1) a psychological approach with
emphasis on the intervening mechanisms between a stressor and its long-term effect and 2) a biological approach
with emphasis on the immediate physiological reaction between stressor and effect. These approaches are
complementary and several models integrated the two approaches.
Representative for these approaches are the models are the Lazarus cognitive approach to stress and Ursins
psychobiological approach to stress.
The cognitive stress model of Lazarus distinguished several phases in the stress process: via primary and
secondary appraisal people make a judgement whether there is a threat and whether they can control it. Based on
this appraisal process they choose a suitable coping strategy. These strategies can roughly be subdivided into
active and passive coping. The outcome of this is dependent on the type of stressor, but in general it shows that
avoidance enhances symptoms while problem oriented coping in many cases results in a perceived reduction of
stress and symptoms. The model is transactional in the sense that the outcome leads to reappraisal of the
exposure.
In the diagram below (Figure 7.2) this process in schematized, taking environmental noise as example.
Figure 7.2. Example of cognitive stress model of Lazarus.
In the model developed by Ursin and his team, stimulus and response expectancy play a key role. Ursin based this
model on earlier work among pilots and parachute jumpers. In this model the person environment interaction and
the concept of homeostasis are central. Stimulus and response expectancy (comparable with primary and
secondary appraisal in the Lazarus and Folkman approach) predict the level of arousal (autonomic and endocrine)
and either lead to restoration of homeostasis or to sustained activation. Sustained activation is associated with
subjective health co plaints and unexplained symptoms.
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Stress as healthy alarm. 7.3.1
Originally the Ursin model was referred to as psychobiological but in more recent work the model is described as
the Cognitive Activation Theory of Stress (CATS) (Ursin and Eriksen, 2004, Ursin and Eriksen, 2010). CATS (see
Figure 7.3) has been developed and tested with regard to stressful work environments (Eriksen HR, 2002,
Kristenson et al., 2004, Ree et al., 2014), but has been also applied to specific research field such as
environmental noise (van Kamp, 1990, Klæboe, 2011) (using a predecessor of CATS combined with Lazarus’
stress model). According to CATS, the stress response results from a perceived “discrepancy between what is
expected to be ‘normal’ and what is happening in reality (actual value)” (Ursin et al., 2004). This perceived
discrepancy (i.e. the stressor) is assumed to induce neurophysiological activation. Neurophysiological activation
due to this discrepancy remains elevated until the individual has succeeded in reducing the disparity between
expected (feared or desired) and actual values (of personal noise exposure, for example). If the difference between
expected and actual values persists, arousal sustains and induces pathophysiological processes. In CATS, “the
real concern is sustained arousal occurring when there is no solution” (Ursin et al., 2010), as the organism is vainly
struggling for homeostasis by trying to exert control on expected and/or actual values. The higher the individual
rates her or his chances to reduce or remove the difference between expected and actual values, the more likely it
is for the organism to return to the arousal level observed prior to the activation. As mentioned before two concept
are key: stimulus expectancy and outcome expectancy. The stimulus expectancy arises from the perceived
probability of an event associated with a specific stimulus while outcome expectancy refers to the perceived
relationship between actions (behaviours) and their results (outcome): positive effects, no control, and negative
effects. A positive outcome expectancy is comparable with active coping while a negative outcome expectancy is
related to avoidance or defence. According to the authors the model has explanatory power in epidemiology,
prevention and treatment of "subjective health complaints". Since the model is primarily a model on work stress it is
fit to be applied to the situation of flight attendants and pilots.
Figure 7.3. Schematic presentation of the CATS model (van Kamp, 1990)
Definition of somatically unexplained physical symptoms 7.3.2
The diagnosis of somatically (partly) unexplained physical symptoms is the most prevailing diagnosis in medical
practice. We speak of somatically unexplained physical symptoms when the symptoms exist over several weeks
and adequate tests have not resulted in a sufficient explanation for the symptoms. The diagnosis is however often
made at a later stage or patients are often not properly informed about the diagnosis. Symptoms such as fatigue,
headache, sleep difficulties, gastrointestinal disturbances and musculoskeletal pain are often referred to as non-
specific because they are very common in the general population, occur in multiple organ systems and can be
caused by a variety of factors, sometimes unknown (Yzermans et al., 2016). In general practice e.g. at least 50% of
the presented complaints are not explained by organ pathology. A whole range of terms has been suggested to
refer to these symptoms (for review see Creed et al. (2009). Symptoms attributed to environmental factors form a
subcategory within the domain of somatically unexplained physical symptoms. Although there might be a causal
foundation for the attributions, as in the case of contaminated cabin air in aircraft, it is often concluded that there is
no scientific base for a causal mechanism, given the actual low exposure levels. Within the broader domain of
symptoms attributed to environmental factors again several subgroups are discerned, related to different types or
sources of exposures such as multi chemicals, indoor climate, (low frequency) noise, electromagnetic fields and
food additives. In some cases not the symptoms obtain a key role, but the assumed sensitivity behind them such
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as noise sensitivity, multi-chemical sensitivity, electro sensitivity and more broadly: environmental sensitivity. This
genetic and/or acquired sensitivity might then explain why some people suffer from complaints after exposure to
low doses of some toxic agent while others do not.
Recent research (Baliatsas, 2015) comparing symptom patterns between different (self-declared) groups of
sensitives versus non sensitives showed considerable overlap between self-reported as well as GP registered
symptoms, but also specific patterns related to organ systems. For example multi chemical sensitives scored
higher on respiratory and gastrointestinal symptoms, while electro-hypersensitive people scored higher on
cardiovascular symptoms and symptoms related to the psychological and neuro-vegetative function system.
Prevalence 7.3.3
Here we briefly discuss what is known about the prevalence of health problems and symptoms in cabin crew and in
the general population and subgroups. In the specific literature on symptoms in cabin crew we make a distinction
between relatively large and small epidemiological studies and case reports. This overview has by no means the
pretention of completeness and rather serves as an indication for the type of health complaints reported by cabin
crew while using prevalence figures in the general population (and its subgroups) as reference.
Derived from a relatively large epidemiological study
McNeely et al (2014) made a comparison between the self-reported health of US flight attendants (pilots were not
included) and the general population. The association of symptoms with exposure to the broad aircraft
environmental was also explored. Job tenure was used as proxy of exposure. Data from a survey among flight
attendants of two large domestic US airlines gathered in 2007 were compared with data obtained from surveys in a
cohort in the framework of the National Health and Nutrition study (NHANES) over the period between 2005 and
2008 adjusting for age, gender, education, smoking and body mass index (BMI). Moreover, only participants from
the general population were selected who matched with the flight attendants in terms of income, level of education
and current employment (note: as indicators of social economic status). The response rate was 48% bringing the
number of participants to 2.613, completed by an additional 1.398 surveys in attendants of the same airlines.
Health status was inventoried in a broad way. Our focus is on somatically unexplained physical symptoms only.
Also at the exposure side a broad range of conditions is considered which are characteristic for current work
circumstances of flight attendants. These include disrupted circadian rhythm, physical demands in restricted cabin
quarters, cosmic radiation, air contaminants, noise, vibration, low pressure and humidity and gravitational forces.
Longer tenure implies longer exposure to these potential hazards. Frequent acute symptoms in the flight attendants
include symptoms related to different organ systems such as sinus congestion, bloating, anxiety, and
musculoskeletal symptoms prevailing in 23 – 29% of flight attendants. From the longer term symptoms, the
respiratory symptoms (sinusitis, allergies and reactive airways) score by far the highest (54%), followed by general
fatigue (37%), joint aches and pains (33%) and severe headaches (23%).
The inventoried symptoms among flight attendants only partly overlap with those from the NHANES study.
Prevalence was compared by means of the standardized prevalence rate (SPR), weighted by age and analyzed
separate per gender. With the approach expected prevalences are derived from the data in the general population
and compared with the observed prevalences in the flight attendant group. This comparison revealed an increase
in chronic bronchitis, with prevalences of 3.6 versus 13.5% in males and 5.1 and 16.1% in females (SPR: 3.5 and
2.7 respectively). This is of value considering that the number of smokers is considerable lower in the flight
attendants. Sleep disorders are quite prevalent in both males as females with prevalences of 32 NS 34% versus 8
and 6% in the general population (SPR’s of 3.5 and 5.5). Fatigue was twice as high in male attendants than in the
general population and symptoms of depression 5 times higher in males and 2 times higher in females (prevalence
of <1 ad 1.5 in the general population and 3.7 in male and 3.8 in female flight attendants. Chronic bronchitis was
shown to be associated with longer job tenure after adjusting for smoking, age, education, and overweight. This is
according to the authors possibly linked to passive smoking( SHTS exposure), with 41% working longer than 20
years. Another explanation could be the cabin air quality, but as stated before, the broad range of symptoms which
could theoretically be attributed to the cabin air quality have not been systematically studied in cabin crew. Other
associations with job tenure found were skin cancer, hearing loss, depression and anxiety, while this was not the
case for sleep disorders and migraines.
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In conclusion: the McNeely study showed that a whole range of health conditions pertaining to different organ
systems (respiratory, neurological, musculoskeletal, gastrointestinal) was higher in flight attendants as compared to
the general population and some of these show an association with job tenure.
Derived from smaller epidemiological studies and case reports
Reviews of studies on flight attendants health commonly conclude that most studies are not based on random
samples, outdated while the industry is changing rapidly, based solely on self- reported data and suffer from low
response rates (Nagda et al., 2003, Griffiths et al., 2012, McNeely et al., 2014).
In the framework of this project again a systematic literature review was performed (see section 3) including a set
of smaller epidemiological studies and case reports among pilots and flight attendants.
In appendix 1 and 2,the findings regarding symptom report among aircraft crew are summarized. In the following
paragraph these findings are compared with what was reported by McNeely and symptom patterns in the general
population.
The 19 included studies consisted of 3 case control studies,11 case studies and 5 small epidemiological studies.
Although most studies are small, often not representative and primarily based on self-reported symptoms, they give
good insight into the symptoms people attribute to cabin air quality and how these relate to those found a the larger
epidemiological study discussed above and in the general population. The most frequently reported symptom is
headache which shows up in 11 out of 19 studies with prevalence estimates ranging between 15-86%, followed by
cognitive functioning in 8 studies with rates between 25-78% and dizziness and disorientation also in 8 studies (8-
72%). Nausea, fatigue/exhaustion and irritated eye show up in 7 studies with again a broad range of estimated
prevalences 29-58%, 48-78% and 14-76% respectively. Other symptoms including respiratory complaints were
only mentioned in 2-4 studies, while depression only showed up in 1 study.
In most studies people attributed their symptoms to cabin air quality (fumes and smoke) and in two studies people
26-53% of the participants described themselves as sensitive to multiple-chemicals. As stated before, the quality of
the studies was moderate and suffered from selection bias, low response rates, small sample sizes. The
prevalences reported are highly dependent on the specific participants included and the way they were selected or
self-selected. This makes it hard to allow for any comparison with other data, both from the same professional
groups as in the study of McNeely et al. (2014) and the general population. Even though there is some parallel in
the most prevalent symptoms the patterns is not consistent and the estimated prevalences are in most cases not
comparable and in general higher than in the well-designed epidemiological study among flight attendants. This
confirms again that we are dealing with selection bias. The next paragraph summarizes the main differences.
Background prevalence estimates from different studies
In the Netherlands, the percentage somatically unexplained physical symptoms in outpatient services of somatic
specialists is estimated to be 41 to 66% (Tak and Bax-aan de Stegge, 2014) and 35% in a neurological clinic
(Snijders et al., 2004). It is estimated that in 10-30% of these patients the symptoms become chronic. Haller et al.
(2015) estimates the prevalence among outpatients to be 40-49%. In the general population headaches, belly
aches and fatigue are the most common symptoms with prevalences between 30-35% (Kroenke et al., 1990,
Kroenke, 2003) and 52% (Nimnuan et al., 2001). In an overview of Creed (2009) a summary is given of the
prevalence of symptoms per type of clinic: 30% concerned neurological symptoms, 40% gastrointestinal and 53%
general medicine, which is highly comparable to what Nimnuan found. In flight personnel general fatigue,
headaches, and muscular skeletal complaints come forward as the most common symptoms in general and in
smaller studies with specific groups of participants and case studies after an event, with prevalence estimates
between 48-78%, 15-86%, 23-29%. While respiratory symptoms come forward as the most prevailing in cabin
crew, these symptoms do not show up in the specific case studies. Nausea, and neuropsychological symptoms
such as cognitive impairment, disorientation/dizziness are most often mentioned and attributed to the exposure to
contaminated air in the cabin as well as irritated eyes.
Estimates on the prevalence of psychiatric comorbidity are inconclusive, but in general it is found that the number
of symptoms is a good predictor of psychiatric problems (see also thesis Van Bellis van den Berg (2007)). It is well
known that psychiatric problems are often missed in the group of people who tend to attribute their symptoms to
environmental factors and moreover they are also taboo especially in this patient group. These associations have
not been studied thus far in the specific occupational group of flight personnel.
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7.4 Concluding observations
There is for quite some time concern about the association between the air quality in aircraft (and its broad variety
of compounds) and health complaints typical among pilots and cabin crew concerning respiratory system, mood,
concentration, dizziness etc. These complaints have not been systematically mapped; no causal relation has jet
been established between these prevailing symptoms and exposure. A broad range of symptoms related with
different organ systems has been found among flight personnel, but these have not been systematically studied.
However, overall statements about the prevalence of somatically unexplained physical symptoms are hard to make
because symptoms overlap, estimates are dependent on the definitions used and the participants in the study. This
is true for specific studies in flight attendants and pilots, but also for the international literature on the prevalence of
somatically unexplained physical symptoms. When trying to compare the estimated prevalence of somatically
unexplained physical symptoms, problems concerning comparability in terms of study population, composition of
the sample and symptoms included are encountered.
The main aim of this section was to explore the factors that can contribute to the development of health complaints
caused by exposure to potentially contaminated cabin air and provide potential explanations why a broad range of
(un)specific types of symptoms are reported in only a limited number of individuals exposed.
The complete metabolic pathway and the contribution of interindividual variability in the metabolic enzymes is still
largely unknown for the majority of industrial chemicals, including CACs. Nevertheless, differences in sensitivity
can be expected for compounds that rely on cytochrome P450 enzymes for their metabolism. This may render a
specific subpopulation that could be approximately 1000-fold more sensitive to specific chemicals. The broad range
of compounds in the air in combination with other stressors, typical for working in an aircraft at irregular times has
not been systematically mapped. In view of this great variety in symptoms and the lack of specificity, it cannot be
ruled out that part of the symptoms cannot be explained by actual exposure levels. Whether or not occupational
conditions are responsible for the reported complaints remains unknown until the complete set of potential
chemical exposures is known, including their exposure levels, resulting internal dose levels, full spectrum of
molecular targets (i.e., all different modes of action) and the related no-effect concentrations.
The literature shows that we are dealing with symptoms which are also quite common in the general population
and in primary and secondary health care patients. It has been found that these symptoms do not always profit
from treatment and often become chronic. Key feature is that the symptoms are often attributed to external causes,
This does not necessarily imply a priori knowledge about plausibility of a relation between the symptoms and
reported symptoms.
Preliminary points of attention in case of further epidemiologic research in cabin crew:
• Define relevant symptoms and clear case characterization, possibly distinguish hereby between specific
and non-specific symptoms.
• Systematic study among pilots and flight attendants and control group of these groups of symptoms
• Include context, personality situational factors and pay attention to triggering and maintaining factors as
well.
• Include aspects as perceived threat and control and coping strategies.
• Ideal design would be a combination of survey among specific groups and control group + diary and
provocation study. However, this might be quite problematic because it cannot be done at random
moments during the flight.
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8 Conclusions
The objective of the AVOIL study was to characterize the toxic effects of chemical compounds that are released
into the cabin or cockpits of transport aircraft. The characterisation was aimed at the toxic effects of aviation turbine
engine oil as a mixture of compounds, including potential pyrolysis breakdown products.
From the experimental work on detecting chemicals it is concluded that the commercial oils included in this study
do contain TCP, however no tri-ortho cresyl phosphate isomers could be detected. The overall emission of both oils
differs at both simulations. Due to increase of temperature, more TCPs will emit, however, the mass fraction of the
emitted TCP isomers was comparable to the TCP oil composition. PAHs such as naphthalene are formed probably
due to partial oxidation. VOCs (e.g., 2-hexanone), aldehydes (e.g. formaldehyde and acetaldehyde) were clearly
present at relative high concentrations. The CO and mineral oil concentration increased drastically at cruise
simulation temperature, i.e., 375°C (± 25 °C).
Dedicated work on pyrolysis of the oils with comprehensive GC-MS (GCxGC-MS ) conformed the above formulated
conclusions. A list of 127 compounds was identified under both nitrogen and oxygen conditions in all oils and
during different simulated flight stages. The basic oil patterns of the two tested oils demonstrated no significant
differences. A set of TCP isomers, 4-octyl-N-(4-octylphenyl)-benzenamine and N-phenyl-1-naphthaleneamine
could be identified in the basic oil patterns. TCP isomer peaks were found in all three oils. N-phenyl-1-
naphthaleneamine was only found in oil Bn, although it did not visually appear in the TIC chromatogram, which
indicates a low concentration. Heating under nitrogen led to an increase in the number of compounds found, and
led to the identification of 24 compounds in the vapour, found in all applied oils. A number of compounds was
identified unique for either oil An or oil Bn. In addition, used oil (Au) appeared to contain newly identified compounds
compared to unused oil (An), and a number of compounds originally present appeared to have disappeared during
use in an engine jet. This indicates that during the lifetime of an oil, substantial changes in composition occur.
Based on the study on toxic effects of the oils after pyrolysis it was concluded that the current data indicate that
neuroactive pyrolysis products are present, but that their concentration in the presence of an intact lung barrier is
that low that it could not be appointed as a major concern for neuronal function. Moreover, the non-significant
effects appeared to be transient as neuronal activity following 24h exposure is very comparable to medium
controls. However, prolonged exposure to pyrolysis products may aggravate their potential neurotoxicity. Additional
research may thus need to focus on prolonged and/or repeated exposure to pyrolysis products.
Analysis of the human sensitivity variability factor showed that the complete metabolic pathway and the contribution
of inter individual variability in the metabolic enzymes is still largely unknown for the majority of industrial
chemicals, including CACs. Differences in sensitivity between humans can be expected for compounds that rely on
cytochrome P450 enzymes for their metabolism. This may explain the symptoms observed in a specific
subpopulation of the people with health problems that may be related to cabin air. However, the broad range of
compounds in the air in combination with other stressors, typical for working in an aircraft at irregular times has not
been systematically mapped. Further, in view of the great variety in symptoms and the lack of specificity, it cannot
be ruled out that part of the symptoms cannot be explained by actual exposure to chemicals. Whether or not
occupational conditions are responsible for the reported complaints remains unknown until the complete set of
potential chemical exposures is known, including their exposure levels, resulting internal dose levels, full spectrum
of molecular targets (i.e., all different modes of action) and the related no-effect concentrations. A suggestion for
follow-up research is to define the specific symptoms as reported by flight and cabin crew, in order to investigate if
a syndrome can be defined.
For future work on risk assessment and maximum exposure levels, it needs to be taken into account that the
conditions in cabin air may differ from the standard conditions on which exposure limits are normally based, for
example the air pressure, humidity and longer working hours. These aspects need further consideration. In
addition, also possible effects relating to mixture toxicology need further investigation.
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Appendix 1 Selection of additional literature
Reference Included Justification
https://www.faa.gov/data_research/researc
h/med_humanfacs/oamtechreports/2010s/
media/201520.pdf
Yes Very recent and potentially relevant,
include in analysis category 4
http://www.ohrca.org/wp-
content/uploads/2014/05/Medicalprotocol0
31909.pdf
Yes OHRCA full study report was already
selected for analysis category 1 & 2 as it
focusses on health effects and exposure.
On second inspection it also contains some
new engine oil measurements (page 39)
and will therefore include as well in the
analysis category 4
http://www.ohrca.org/wp-
content/uploads/2014/05/quickreference.pd
f
http://www.ohrca.org/wp-
content/uploads/2014/08/finalreport.pdf
http://www.cdc.gov/niosh/docket/archive/pd
fs/niosh-220/95-117.pdf
No This report contains good practice
guidelines for air sampling in general and is
not within the scope of this literature
review. Therefore it will be excluded from
the analysis.
http://www.iso.org/iso/iso_catalogue/catalo
gue_tc/catalogue_tc_browse.htm?commid
=52702&published=on&includesc=true
No This report contains good practice
guidelines for air sampling in general and is
not within the scope of this literature
review. Therefore it will be excluded from
the analysis.
http://www.iom-
world.org/pubs/IOM_TM1106.pdf
Yes This report was already selected for
analysis of category 2 as it focusses on
exposure assessment.
http://ec.europa.eu/environment/air/quality/
standards.htm
No This report contains air quality limit values
in general and is not within the scope of
this literature review. Therefore it will be
excluded from the analysis.
http://www.airspacemag.com/flight-
today/clearing-the-cabin-air-1-19794696/
No This is an online article about a research
consortium yet to be started and does not
contain any relevant data.
https://dspace.lib.cranfield.ac.uk/bitstream/
1826/5305/1/AircraftCabinAirSamplingStud
yPart1FinalReport%2020110420.pdf
Yes The Cranfield study was already selected
for analysis category 2 as it focusses on
exposure assessment
http://asrs.arc.nasa.gov/docs/rpsts/cabin_fu
mes.pdf
No Narrative description of fire, smoke, fumes
or odor incidences. Not within the scope of
the focused literature review.
http://www.ncbi.nlm.nih.gov/pmc/articles/P
MC4444275/
No The literature review focusses on effects
related to engine oil, fumes and pyrolysis
products. Therefore, effects of ozone are
not taken into account.
http://www.ncbi.nlm.nih.gov/gquery/?term=
cabin+air+quality
No See methodology for details on used
search term
http://www.ncbi.nlm.nih.gov/books/NBK207
470/
Yes Potentially relevant for all four categories,
include in analysis
http://www.ncbi.nlm.nih.gov/books/NBK207
485/pdf/Bookshelf_NBK207485.pdf
CAQ sensing technologies & studies –
state of play
Yes This note relates to the overall cabin air
quality and exposure measurements. It
gives a summary of some exposure
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measurement campaigns focused on
VOCs. It can be included for category 2 as
it focusses on exposure assessment;
depending on the focus of the analysis of
the exposure measurement data.
Source apportionment of volatile organic
compounds (VOCs) in aircraft cabins
No This article focusses on exposure to VOCs
in the cabin and is already discussed in the
summary note above.
In-Flight/Onboard Monitoring: ACER’s
Component for ASHRAE 1262, Part 2
No This article focusses on exposure to VOCs
in the cabin and is already discussed in the
summary note above.
Net in-cabin emission rates of VOCs and
contributions from outside and inside the
aircraft cabin
No This article focusses on exposure to VOCs
in the cabin and is already discussed in the
summary note above.
Measurements of volatile organic
compounds in aircraft cabins. Part II:
Target list, concentration levels and
possible influencing factors
No This article focusses on exposure to VOCs
in the cabin and is already discussed in the
summary note above.
Measurements of volatile organic
compounds in aircraft cabins. Part I:
Methodology and detected VOC species in
107 commercial flights
No This article focusses on exposure to VOCs
in the cabin and is already discussed in the
summary note above.
Professor Michael Bagshaw. (2014). Health
Effects of Contaminants in Aircraft Cabin
Air. Summary Report v2.7
Yes This report was already selected for
analysis of category 1 as it focusses on
health effects. On second inspection it also
contains some theoretical maximum oil
concentrations in the cabin and will
therefore include as well in the analysis
category 2.
Evaluation of shipboard formation
of a neurotoxicant
(trimethylolpropane phosphate)
from thermal decomposition of
synthetic aircraft engine
lubricant
No This article also retrieved with the literature
search but was excluded because it
focusses on shipboard fires
Sensors and Prognostics to Mitigate Bleed
Air Contamination Events 2012 Progress
Report
Yes This article contains an overview of
previous studies on thermal degradation of
aviation engine oil and some new data. Will
be included in analysis.
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Appendix 2 Summarizing the health symptoms reported by cabin crew after exposure to
contaminated bleed air.
Table A.1. Frequency of symptoms recorded for 36 aircrew exposed to contaminated cabin air (Somers 2005) .
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Figure A.1. Data on eye and skin irritation signs and symptoms from 50 exposed aircrew members (BAe 146 and
A320) (Winder et al., 2002).
Figure A.2. Data on respiratory and cardiovascular symptoms from 50 exposed aircrew members (BAe 146 and
A320) (Winder et al., 2002).
Figure A.3. Data on gastrointestinal/renal signs and symptoms from 50 exposed aircrew members (BAe 146 and
A320) (Winder et al., 2002).
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Figure A.4. Data on neuropsychological signs and symptoms from 50 exposed aircrew members (BAe 146 and
A320) (Winder et al., 2002).
Figure A.5. Data on neurological signs and symptoms from 50 exposed aircrew members (BAe 146 and A320)
(Winder et al., 2002).
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Figure A.6. Data on reproductive signs and symptoms from 50 exposed aircrew members (BAe 146 and A320)
(Winder et al., 2002).
Figure A.7. Data on general signs and symptoms from 50 exposed aircrew members (BAe 146 and A320) (Winder
et al., 2002).
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Figure A.8. Numbers of symptoms reported by 39 pilots exposed to contaminated aircraft cabin air (Harper, 2005).
Table A.2. Numbers of symptoms reported by 19 pilots and 2 flight attendants flying on a BAe 146 aircraft (Cox and
Michealis. 2002).
Table A.3. Numbers of symptoms reported by 106 pilots flying on a B737, B757 and A320 aircraft (Michealis,
2003).
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Table A.4. Description of subject and results of examination of the 26 airline attendants (Heuser et al., 2006).
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Table A.5. Symptoms reported by 35 flight crew during 4 months period (van Netten, 1998).
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Table A.6. Symptoms summary of seven case studies (Winder and Balouet, 2001).
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Figure A.9. Symptoms summary of 34 flight crew members (Abou-Donia et al., 2013).
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Table A.7. Health symptoms reported by at least 15% of the flight attendants (OHRCA, 2014).
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Figure A.10. Health symptoms and frequency reported in survey of 640 aircrew by Toxic Free Airlines (EPAAQ,
2011).
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Appendix 3 Order of magnitude of measurements of oil compounds found in a realistic setting in
an aircraft.
Table A.8. OP levels detected in air samples collected during flight (Solbu et al., 2011).
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Table A.9. VOCs detected in air samples collected during 14 domestic flights (Wang et al., 2014)
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Appendix 4 Identification of various compounds in oil vapours (GC/MS/TD)
Compounds were identified based on peak deconvolution with AMDIS followed by a target library search. The
target library contains over a 1000 compounds with spectra and retention indices. The identification was based on
the NET match factor, a combination of the match factor and the retention index, with a minimum NET match factor
of 80. Compounds that were not identified with the target library search were tentatively identified by a search in
the NIST library with a minimum match factor of 80.
Table A.10. Identification of various aldehydes and ketones in oil vapour.
Oil type An An Au Au Bn Bn
Temperature range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Test duration (min) 30 60 30 60 30 60
Compounds CAS
Aldehydes
heptanal 111-71-7 X X X X X
nonanal 124-19-6 X X X X
2-methylpropanal 78-84-2 X X
2-propenal 107-02-8 X X X X X X
2-butenal 4170-30-3 X X X X X
2-pentenal 1576-87-0 X X X X X
trans-2-hexenal 6728-26-3 X X X
2-heptenal 57266-86-1 X X X
2-octenal 2548-87-0 X
2-methyl-2-propenal 78-85-3 X X X X X X
2-methyl-2-butenal 1115-11-3 X X
2-ethylpropenal 922-63-4 X X X X
3-furaldehyde 498-60-2 X X X
Ketones
2-butanone 78-93-3 X X X X X X
2-pentanone 107-87-9 X X X X X X
2-heptanone 110-43-0 X X X X
2-octanone 111-13-7 X
3-hexanone 589-38-8 X X X
3-heptanone 106-35-4 X X X
4-heptanone 123-19-3 X X X
5-nonanone 502-56-7 X X X X
2,3-butanedione 431-03-8 X
2,5-hexanedione 110-13-4 X X X X X
cyclopentanone 120-92-3 X X
3-methylcyclohexanone 591-24-2 X X X
1-buten-3-one 78-94-4 X X X X X
1-penten-3-one 1629-58-9 X X X
1-hexen-3-one 1629-60-3 X X
1-hepten-3-one 2918-13-0 X X
3-penten-2-one 625-33-2 X X X X X
3-hexen-2-one 763-93-9 X X X
5-hexen-2-one 109-49-9 X X X X
2-methyl-2-cyclopenten-1-one 1120-73-6 X X X
1-hydroxy-2-propanone 116-09-6 X X X
1,4-naphthalenedione 130-15-4 X X
1,2-naphthalenedione 524-42-5 X
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Table A.11. Identification of various alkenes in oil vapour.
Table A.12. Identification of various organic acids and esters in oil vapour.
Oil type An An Au Au Bn Bn
Temperature range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Test duration (min) 30 60 30 60 30 60
Compounds CAS
Alkenes
1-pentene 109-67-1 X X X X X
1-hexene 592-41-6 X X X X X
1-heptene 592-76-7 X X X
1-octene 111-66-0 X X X X X
trans-2-hexene 4050-45-7 X X X X
trans-2-heptene 14686-13-6 X X X
cis-2-hexene 7688-21-3 X X X
2-methyl-1-pentene 763-29-1 X X X X
4,4-dimethyl-1-pentene 762-62-9 X X X
2,4,4-trimethyl-1-pentene 107-39-1 X X
cyclopentene 142-29-0 X
cyclohexene 110-83-8 X X
1-methylcyclopentene 693-89-0
1-ethylcyclopentene 2146-38-5 X X X
1-methylcyclohexene 591-49-1 X X X
Oil type An An Au Au Bn Bn
Temperature range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Test duration (min) 30 60 30 60 30 60
Compounds CAS
Organic acids
acetic acid 64-19-7 X X X X
formic acid 64-18-6 X X
propanoic acid 79-09-4 X X X
butanoic acid 107-92-6 X X X
pentanoic acid 109-52-4 X X X
hexanoic acid 142-62-1 X
heptanoic acid 111-14-8 X X X X
octanoic acid 124-07-2 X X X X
decanoic acid 334-48-5 X X X
2-methylbutanoic acid 116-53-0 X X
3,5,5-trimethylhexanoic acid 3302-10-1 X X X
2-propenoic acid 79-10-7 X X
3-butenoic acid 625-38-7 X
Esters
acetic acid, methyl ester 79-20-9 X X X X X
pentanoic acid methyl ester 624-24-8 X X X X X
heptanoic acid methyl ester 106-73-0 X X X X
octanoic acid methyl ester 111-11-5 X
decanoic acid, methyl ester 110-42-9 X X X
2-methylbutanoic acid, methyl ester 868-57-5 X
actic acid, propyl ester 109-60-4 X X
pentanoic acid, 2-propenyl ester 6321-45-5 X X X X
acetic acid, ethenyl ester 108-05-4 X X X
2-propenoic acid, methyl ester 96-33-3 X X X X
3-butenoic acid, methyl ester 3724-55-8 X X X
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Table A.13. Identification of various alkenes in oil vapour.
Oil type An An Au Au Bn Bn
Temperature range (˚C) 20-350 375±25 20-350 375±25 20-350 375±25
Test duration (min) 30 60 30 60 30 60
Compounds CAS
Alcohols
tert-butanol 75-65-0 X X X
2-propen-1-ol 107-18-6 X
Furanes
furan 110-00-9 X X X X
2-methylfuran 534-22-5 X X X X
2-ethylfuran 3208-16-0 X X
2,5-dimethylfuran 625-86-5 X X X
2-ethyl-5-methylfuran 1703-52-2 X X X
2-n-propylfuran 4229-91-8 X
tetrahydrofuran 109-99-9 X X X X X
2-methoxytetrahydrofuran 13436-45-8 X X X
Various components
trans-1,2-dimethylcyclopentane 822-50-4 X X X X X
tetrahydro-2-methylpyran 10141-72-7 X X X X X
methyl 2-oxopropanoate 600-22-6 X X X
phthalic anhydride 85-44-9 X X X
trans-2,3-dimethyl oxirane 21490-63-1 X X X
N-phenylformamide 103-70-8 X X
o-isopropenyltoluene 7399-49-7 X
3- or 4-methylphenol 108-39-4 X X X X X
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Appendix 5 2D TIC chromatogram of TENAX samples
2D TIC chromatogram of TENAX samples obtained from pyrolysis of the oils in the various flight phases (Horizontal
plots respectively Tax, Climb, Cruise, Descent) and under inert and oxygenated conditions (Vertical plots; N2
duplicate and O2). Each plot shows the peak height shown by color gradient against the: 1st dimension time (sec,
x-axis) and 2nd dimension time (sec, y-axis)
Figure A.10: Oil Au
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Figure A.11: Oil An
Figure A.12: Oil Bn
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Appendix 6 Classification of chemicals list
The following table describes the hazard profile of the list of compounds (Table 5.6) identified under both nitrogen
and oxygen conditions, in all oils and during different flight stages. The hazard profile, or classification, of a
substance is an indication of its intrinsic toxicity. A substance is classified, e.g. assigned a notation for a specific
toxicity, according to an internationally agreed classification criteria. The classification can distinguishes between
the various types of toxicity (reprotoxic, carcinogenic, irritant), the potency (4 (less potent) =>1 (most potent), the
frequency needed to exert the toxicity (Single Exposure (SE) vs Repeated Exposure (RE)) and the level of
evidence (1A, 1B and 2) available (for more information, see the Guidance on the criteria for Classification and
Labelling under REACH).
In most cases, the manufacture or importer classifies the substance without any review by an independent
organisation, the so-called self-classification. Different manufacturers classify the same substance differently.
Harmonized classification means the classification is agreed upon in a scientific committee and that classification is
mandatory for every manufacturer or importer.
The substances are run through the Classification and Labelling database of the European Chemical Agency
(ECHA) database. This database contains all classification and labelling information on notified and registered
substances received from manufacturers and importers in the European Union. Manufacturers and importers need
to notify a substance to the Classification and Labelling (C&L) Inventory if they intent to place the substance on the
EU market.
Compound # Name CAS
Harmonized
classification
Self-
classification*
1 Diethyl Phthalate 84-66-2 NC
2 1-Nonene, 4,6,8-trimethyl- 54410-98-9
3 2-Ethylhexyl salicylate 118-60-5 Skin Irrit. 2
4 Acetophenone 98-86-2
Acute Tox. 4
Eye Irrit. 2
5 Benzaldehyde 100-52-7 Acute Tox. 4
6 Benzene, 1,3-bis(1,1-dimethylethyl)- 1014-60-4 NR NR
7 Heptane, 4-methyl- 589-53-7
Asp. Tox. 1
Skin Irrit. 2
STOT SE 3
8 Nonanal 124-19-6 NC
9 2,4-Dimethyl-1-heptene 19549-87-2 Asp. Tox. 1
10 Decanal 112-31-2 Eye Irrit. 2
11 Dodecanoic acid, isooctyl ester 84713-06-4 NC
12 Heptadecane, 2,6,10,14-tetramethyl- 18344-37-1 NR NR
13 Octanal 124-13-0
Skin Irrit. 2
Eye Irrit. 2
14 Dodecane, 4,6-dimethyl- 61141-72-8 NR NR
15 Heptane 142-82-5
Asp. Tox. 1
Skin Irrit.2
STOT SE 3
16 5,9-Undecadien-2-one, 6,10-dimethyl- 689-67-8 Skin Irrit. 2
17 Benzene 71-43-2
Asp. Tox. 1
Skin Irrit. 2
Eye Irrit. 2
Muta. 1B
Carc. 1A
STOT RE 1
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Compound # Name CAS
Harmonized
classification
Self-
classification*
18 Glycidol 556-52-5
Acute Tox. 4
Skin Irrit. 2
Eye Irrit. 2
Acute Tox. 3
STOT SE 3
Muta. 2
Carc. 1B
Repr. 1B
19 Nonane, 2,6-dimethyl- 17302-28-2 NR NR
20 2-Propanol, 2-methyl- 75-65-0
Eye Irrit. 2
Acute Tox. 4
STOT SE 3
21 Decane, 2,3,5,8-tetramethyl- 192823-15-7 NR NR
22 Nonane 111-84-2 Eye Irrit. 2
23 Octane 111-65-9
Asp. Tox. 1
Skin Irrit. 2
STOT SE 3
24 Phenol, 2,4-bis(1,1-dimethylethyl)- 96-76-4
Acute Tox. 4
Skin Irrit. 2
Eye Dam. 1
25 2,5-Hexanediol, 2,5-dimethyl- 110-03-2 Eye Dam. 1
26 Acetic acid, octadecyl ester 822-23-1 NR NR
27 Undecane 1120-21-4 Asp. Tox. 1
28 2-Heptanone, 4-methyl- 6137-06-0 NR NR
29 Hexane, 2,3,4-trimethyl- 921-47-1 NR NR
30
1,2-Benzenedicarboxylic acid, di-2-
propenyl ester 131-17-9
Acute Tox. 4
31 1-Iodo-2-methylundecane 73105-67-6 NR NR
32 Isopropyl Palmitate 142-91-6 NC
33 n-Decanoic acid 334-48-5
Skin Irrit. 2
Eye Irrit. 2
34 Tridecane 629-50-5 Asp. Tox. 1
35 TCP isomer 563-04-2 Acute Tox. 4
36 1-Propanol, 2-methyl- 78-83-1
Skin Irrit. 2
Eye Dam. 1
STOT SE 3
37 1-Tridecanol 112-70-9 NC
38 5-Hepten-2-one, 6-methyl- 110-93-0 Eye Irrit. 2
39 Decane 124-18-5 Asp. Tox. 1
40 Pentane 109-66-0
Asp. Tox. 1
STOT SE 3
41 Pentanoic acid, methyl ester 624-24-8 NC
42 Amylene Hydrate 75-85-4
Skin Irrit. 2
Acute Tox. 4
STOT SE 3
43 Glycerin 56-81-5 NC
44 Heptanal 111-71-7
Skin Irrit. 2
Eye Irrit. 2
45 Heptanoic acid, methyl ester 106-73-0 Skin Irrit. 2
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Compound # Name CAS
Harmonized
classification
Self-
classification*
46 Octane, 3,5-dimethyl- 15869-93-9 NC
47 Phenol 108-95-2
Acute Tox. 3
Skin Corr. 1B
Muta. 2
STOT RE 2
48 Propane, 2-ethoxy-2-methyl- 637-92-3 STOT SE 3
49
1,2-Benzenedicarboxylic acid, bis(2-
methylpropyl) ester 84-69-5
Repr. 1B
50 2-Butanone 78-93-3
Eye Irrit. 2
STOT SE 3
51 Butylated Hydroxytoluene 128-37-0 NC
52 Decanoic acid, 2-ethylhexyl ester 73947-30-5 NC
53 Diazene, dimethyl- 503-28-6 NC
54 Dodecane, 2-methyl- 1560-97-0 NR NR
55 Methacrolein 78-85-3
Acute Tox. 3
Skin Corr. 1B
Eye Dam. 1
Acute Tox. 2
56 Pentadecane 629-62-9 Asp. Tox. 1
57 Benzaldehyde, 4-methyl- 104-87-0
Acute Tox. 4
Eye Irrit. 2
58 Phenol, 3-methyl- 108-39-4
Acute Tox. 3
Skin Corr. 1B
59 2H-Pyran-2-one, tetrahydro- 542-28-9 Eye Dam. 1
60 Benzene, (1,1,2-trimethylpropyl)- 26356-11-6 NR NR
61 Cyclopropyl carbinol 2516-33-8
Acute Tox. 4
Eye Irrit. 2
62 Hexadecen-1-ol, trans-9- 64437-47-4 NR NR
63 Hexanal 66-25-1 Eye Irrit. 2
64 Isobutane 75-28-5 NC
65 Octanoic acid, methyl ester 111-11-5 Skin Sens. 1
66 (2-Aziridinylethyl)amine 4025-37-0 NR NR
67
1,4-Dioxane-2,5-dione, 3,6-dimethyl-,
(3S-cis)- 4511-42-6
Eye Irrit. 2
68 1-Octene, 3,7-dimethyl- 4984-01-4 NC
69 2-Butene 107-01-7 NC
70 Cyclobutylamine 2516-34-9 Skin Corr. 1B
71 n-Hexadecanoic acid 57-10-3 NC
72 Tridecane, 3-methyl- 6418-41-3 NR NR
73 1-Octene 111-66-0 Asp. Tox. 1
74 2(5H)-Furanone, 3-methyl- 22122-36-7
Skin Irrit. 2
Eye Irrit. 2
STOT SE 3
75 Pentanal 110-62-3
Skin Sens. 1
Eye Irrit. 2
Acute Tox. 4
STOT SE 3
76
1,2-Benzenedicarboxylic acid, mono(2-
ethylhexyl) ester 4376-20-9
Skin Irrit. 2
Eye Irrit. 2
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Compound # Name CAS
Harmonized
classification
Self-
classification*
77 2,5-Furandione, dihydro-3-methyl- 4100-80-5
Skin Irrit. 2
Eye Irrit. 2
STOT SE 3
78 Benzeneacetaldehyde 122-78-1
Acute Tox. 4
Skin Sens. 1
79 Dodecanoic acid 143-07-7 Eye Dam. 1
80 Methylglyoxal 78-98-8
Acute Tox. 4
Skin Sens. 1B
Eye Dam. 1
Muta. 2
81 Pentadecane, 2,6,10-trimethyl- 3892-00-0 NR NR
82 2-Hexanone 591-78-6
STOT SE 3
Repr. 2
STOT RE 1
83 Hydroxyurea 127-07-1
Muta. 1B
Repr. 2
84 (S)-2-Hydroxypropanoic acid 79-33-4
Skin Irrit. 2
Eye Dam. 1
85 1,3,5,7-Cyclooctatetraene 629-20-9
Asp. Tox. 1
Skin Irrit. 2
Eye Irrit. 2
STOT SE 3
86 2-Propanone, 1-hydroxy- 116-09-6 NC
87 Butyrolactone 96-48-0
Acute Tox. 4
Eye Dam. 1
STOT SE 3
88 Cyclopentanone 120-92-3
Skin Irrit. 2
Eye Irrit. 2
89 Dodecane 112-40-3 Eye Irrit. 2
90 Eicosane 112-95-8 NC
91 Heptadecane, 2,6-dimethyl- 54105-67-8 NR NR
92 l-Pantoyl lactone 5405-40-3 Eye Dam. 1
93 Pentanoic acid 109-52-4 Skin Corr. 1B
94 Phthalic anhydride 85-44-9
Acute Tox. 4
Skin Irrit. 2
Skin Sens. 1
Eye Dam. 1
Resp. Sens. 1
STOT SE 3
95 trans-3-Decene 19150-21-1 NR NR
96 Undecanal 112-44-7 Skin Irrit. 2
97 1-Hexene 592-41-6 Asp. Tox. 1
98 1H-Indene, 1-methylene- 2471-84-3 NR NR
99 1-Pentene 109-67-1 NC
100 Acetone 67-64-1
Eye Irrit. 2
STOT SE 3
101 Decane, 3,7-dimethyl- 17312-54-8 NR NR
102 Formamide, N-methyl- 123-39-7
Acute Tox. 4
Repr. 1B
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Compound # Name CAS
Harmonized
classification
Self-
classification*
103 Hexane 110-54-3
Asp. Tox. 1
Skin Irrit. 2
STOT SE 3
Repr. 2
STOT RE 2
104 Octane, 1-chloro- 111-85-3 Asp. Tox. 1
105 1,3,5-Cycloheptatriene 544-25-2
Asp. Tox. 1
Acute Tox. 3
Skin Irrit. 2
Eye Irrit. 2
STOT SE 3
106 1-Hexadecanol 36653-82-4 NC
107 3-Undecene, (Z)- 821-97-6 NR NR
108 Benzonitrile 100-47-0 Acute Tox. 4
109 Cyclooctane, 1,4-dimethyl-, cis- 13151-99-0 NR NR
110 Dodecane, 2,7,10-trimethyl- 74645-98-0 NR NR
111 Heptane, 3-ethyl- 15869-80-4 NR NR
112 1-Pentene, 2,4,4-trimethyl- 107-39-1 NC
113 1-Pentene, 2-methyl- 763-29-1 NC
114 1-Propene, 2-methyl- 115-11-7 NC
115 2(3H)-Furanone, dihydro-5-methyl- 108-29-2 NC
116 2-Heptanone 110-43-0 Acute Tox.4
117 2-Hexenal 505-57-7
Acute Tox. 4
Acute Tox. 3
Skin Sens. 1
118 2-Pentanone 107-87-9
Acute Tox. 4
Eye Irrit. 2
STOT SE 3
119 2-tert-Butyltoluene 1074-92-6 NC
120 Acetic acid 64-19-7 Skin Corr. 1A
121 cis-2-Nonene 6434-77-1
Asp. Tox. 1
Skin Irrit. 2
Eye Irrit. 2
STOT SE 3
122 Decane, 1-chloro- 1002-69-3 Carc. 2
123 Heptanoic acid 111-14-8 Skin Corr. 1B
124 Isopropyl Myristate 110-27-0 NC
125 Tetradecanoic acid 544-63-8 NC
126 1-Pentene, 4-methyl- 691-37-2
Asp. Tox. 1
Or
Skin Irrit. 2
Eye Irrit. 2
STOT SE 3
127 2-Cyclopenten-1-one 930-30-3 NC
* according to the largest number of notifiers
NC = not classified for human health effects
NR = not registered under REACH
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Acute Tox = Acute toxicity
Asp. Tox. = Aspiration toxicity
Eye Irrit. = Eye irritation
Eye Dam. = Eye Damage
Skin Irrit. = Skin irritation
Skin Corr. = Skin corrosion
Skin Sens. = Skin sensitisation
Resp. Sens = Respiratory sensitisation
STOT SE = Specific Target Organ Toxicity Single
Exposure
STOT RE = Specific Target Organ Toxicity Repeated
Exposure
Muta. = Mutagenicity
Carc. = Carcinogenicity
Repr.= Reprotoxicity
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Appendix 7 Chemical analysis of the vapour generated for the in vitro exposures.
Sample description
Generation into air-liquid interface
Generation into air-liquid interface
Generation into air-liquid interface
Generation into air-liquid interface
Oil type Oil An Oil An Oil Bn Oil Bn
Component µg/m3 (oil vapor) µg/m
3 (oil vapor) µg/m
3 (oil vapor) µg/m
3 (oil vapor)
TiPP 0,03 0,02 0,06 0,03 TBP 0,90 0,72 0,74 0,37 TCEP 0,04 0,04 0,03 0,03 TCPP-1 0,05 0,08 0,05 < DBPP < 0,02 < < TPhP 0,02 0,03 0,02 0,02 DCP-1 0,05 < 0,02 < DCP-2 0,04 < 0,02 < T(m,m,m)CP 3,3 2,6 5,7 4,6 T(m,m,p)CP 7,4 5,6 15 11 T(m,p,p)CP 6,5 5,1 13 9,8 T(p,p,p)CP 1,9 1,5 3,4 2,5
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Appendix 8 Abbreviations
ADSE : Aircraft Development and Systems Engineering AMDIS
:
Automated Mass Spectral Deconvolution and Identification System
An : New oil of brand A ASE : Accelerent solvent extraction ASTM : American Society for Testing and Materials
Au : Used oil of brand A AVOIL : Aviation oil
Bn : New oil of brand B CO : Carbon monoxide DCM : Dichloromethane DNPH : Dinitrophenylhydrazine FTIR : Fourier transform infrared spectroscopy GC-FID : Gaschromatography-Flame ionization detector IRAS : Instritute for Risk Assesment Sciences ISO : International Standardisation Organisation NEN : Dutch Normalisation Institute NEN-EN : Dutch Standard-European Standard NIOSH : National Institute for Occupational Safety and Health NIST : National Institute of Standards and Technology NM : Nautical miles NRC : National Research Council OPCW : Organisation for the prohibition of chemical wapons OPE : Organo Phosphate Esters PAH : Polycyclic Aromatic Hydrocarbons PNC : Particle number concentrations RIVM : National Institute for Public Health and the
Environment TCN : Tetrachloro naphthalene TCP : Tricresyl phosphate TMPP : Trimethylolpropane phosphate TNO : Netherlands organisation for Applied Science ToF-MS : Time-of-Flight Mass Spectrometer UME : Unidentified complex mixtures VOC : Volatile Organic Compounds VU : Vrije Universiteit Free University
Konrad-Adenauer-Ufer 350668 CologneGermany